Engineered microbe-targeting molecules and uses thereof

ABSTRACT

Described herein are engineered microbe-targeting or microbe-binding molecules, kits comprising the same and uses thereof. Some particular embodiments of the microbe-targeting or microbe-binding molecules comprise a carbohydrate recognition domain of mannose-binding lectin, or a fragment thereof, linked to a portion of a Fc region. In some embodiments, the microbe-targeting molecules or microbe-binding molecules can be conjugated to a substrate, e.g., a magnetic microbead, forming a microbe-targeting substrate (e.g., a microbe-targeting magnetic microbead). Such microbe-targeting molecules and/or substrates and the kits comprising the same can bind and/or capture of a microbe and/or microbial matter thereof, and can thus be used in various applications, e.g., diagnosis and/or treatment of an infection caused by microbes such as sepsis in a subject or any environmental surface. Microbe-targeting molecules and/or substrates can be regenerated after use by washing with a low pH buffer or buffer in which calcium is insoluble.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C §119(e) of U.S.Provisional Application Nos. 61/508,957 filed Jul. 18, 2011; 61/605,081filed Feb. 29, 2012; and 61/605,052 filed Feb. 29, 2012, the contents ofwhich are herein incorporated by reference in their entirety.

GOVERNMENT SUPPORT

This invention was made with Government support under grant no.N66001-11-1-4180 awarded by DARPA. The Government has certain rights inthe invention.

TECHNICAL FIELD

Described herein relates generally to molecules, products, kits andmethods for detecting and/or removing microbes in a sample or a targetarea, including bodily fluids such as blood and tissues of a subject,food, water, and environmental surfaces.

BACKGROUND

Sepsis is a major cause of morbidity and mortality in humans and otheranimals. In the United States, sepsis is the second leading cause ofdeath in intensive care units among patients with non-traumaticillnesses. It is also the leading cause of death in young livestock,affecting 7.5-29% of neonatal calves, and is a common medical problem inneonatal foals. Despite the major advances of the past several decadesin the treatment of serious infections, the incidence and mortality dueto sepsis continues to rise.

Sepsis results from the systemic invasion of microorganisms into bloodand can present two distinct problems. First, the growth of themicroorganisms can directly damage tissues, organs, and vascularfunction. Second, toxic components of the microorganisms can lead torapid systemic inflammatory responses that can quickly damage vitalorgans and lead to circulatory collapse (i.e., septic shock) and, oftentimes, death.

Sepsis is a systemic reaction defined by the American College of ChestPhysicians and the Society of Critical Care Medicine by a systemicinflammatory response (SIRS) in response to a confirmed infectiousprocess. SIRS is defined by the presence of two or more of thefollowing: altered body temperature (<36° C. or >38° C.), tachycardia(heart rate >90/min), tachypnea (respiratory rate >20/min) or hypocapnia(P_(a)CO₂ less than 4.3 kPa), leucopenia (white blood cells (WBCs)<4000cells/mm³ or leucocytosis (>12000 WBC/mm³) or >10% band forms. Theconfirmation of the infectious process is confirmed by microbiologicalmeans (stain, culture, antigenemia or antigenuria, nucleic aciddetection) or pathognomonic signs of infection obtained by imaging orclinical examination. The infection can affect any organ system, but themore severe cases present as septicemia (i.e., organisms, theirmetabolic end-products or toxins in the blood stream), bacteremia (i.e.,bacteria in the blood), toxemia (i.e., toxins in the blood), endotoxemia(i.e., endotoxin in the blood). Sepsis can also result from fungemia(i.e., fungi in the blood), viremia (i.e., viruses or virus particles inthe blood), and parasitemia (i.e., helminthic or protozoan parasites inthe blood). Thus, septicemia and septic shock (acute circulatory failureresulting from septicemia often associated with multiple organ failureand a high mortality rate) may be caused by various microorganisms.

There are three major types of sepsis characterized by the type ofinfecting organism. For example, gram-negative sepsis is the mostfrequently isolated (with a case fatality rate of about 35%). Themajority of these infections are caused by Escherichia coli, Klebsiellapneumoniae and Pseudomonas aeruginosa. Gram-positive pathogens such asthe Staphylococci and Streptococci are the second major cause of sepsis.The third major group includes fungi, with fungal infections causing arelatively small percentage of sepsis cases, but with a high mortalityrate; these types of infections also have a higher incidence inimmunocomprised patients.

Some of these infections can be acquired in a hospital setting and canresult from certain types of surgery (e.g., abdominal procedures),immune suppression due to cancer or transplantation therapy, immunedeficiency diseases, and exposure through intravenous catheters. Sepsisis also commonly caused by trauma, difficult newborn deliveries, andintestinal torsion (especially in dogs and horses). Infections in thelungs (pneumonia), bladder and kidneys (urinary tract infections), skin(cellulitis), abdomen (such as appendicitis), bone (osteomyeltitis) andjoints (arthritis) and other areas (such as meningitis) can spread andalso lead to sepsis. In some circumstances, ingestion ofmicrobe-contaminated water, fluid or food, or contact withmicrobe-covered environmental surfaces can cause infections that lead tosepsis, and infection with food-borne and water-borne pathogens such asShigella spp, or certain serotypes of Escherichichia coli (such asO157H7), Salmonella spp including Salmonella enterica serovar typhi orListeria monocytogenes can also lead to sepsis.

Many patients with septicemia or suspected septicemia exhibit a rapiddecline over a 24-48 hour period. It has been reported that patientswith septic shock require adapted treatment in less than 6 hours inorder to benefit from antimicrobial therapy. Thus, rapid and reliablediagnostic and treatment methods are essential for effective patientcare. Unfortunately, a confirmed diagnosis as to the type of infection,e.g., sepsis, traditionally requires microbiological analysis involvinginoculation of blood cultures, incubation for 18-24 hours, plating thecausative microorganism on solid media, another incubation period, andfinal identification 1-2 days later. Even with immediate and aggressivetreatment, some patients can develop multiple organ dysfunction syndromeand eventually death. Hence, there remains a strong need for improvedtechniques for diagnosis and treatment of patients with infectiousdiseases, blood-borne infections, sepsis, or systemic inflammatoryresponse syndrome. The ability to rapidly detect infectious pathogens infood, water, and/or environmental surfaces would also have great valuefor preventing infections and sepsis in the population.

SUMMARY

Embodiments described herein are based on, at least in part, engineeringa microbe-targeting molecule or a microbe-binding molecule. For example,in one embodiment, a microbe-targeting molecule is engineered by fusingthe carbohydrate recognition domain and neck region of acarbohydrate-binding protein (e.g., mannose-binding lectin) to theC-terminal of a Fc fragment of human IgG1. Such microbe-targetingmolecules can be also modified to reduce the complement activation andcoagulation side effects which are present in the wild-typemannose-binding lectin, and can complicate binding and detection.Further, the microbe-targeting molecules described herein can beengineered, e.g., by inserting an AKT tripeptide to the N-terminal ofthe Fc fragment for site-specific biotinylation, such that theircarbohydrate recognition domains orient away from a substrate to whichthey attach, thus increasing the microbe-binding capacity. Themicrobe-targeting molecules can be attached to various substrates, e.g.,a magnetic microbead, in a multivalent oriented manner to form amicrobe-targeting substrate. The term “microbead” as used hereingenerally refers to a bead or a particle of any material having a sizeof about 0.001 μm to about 1000 μm or about 0.001 μm to about 100 μm, orabout 0.01 μm to about 10 μm. In one embodiment, the microbead is ananobead. The term “nanobead” as used herein generally refers to a beador particle having a size ranging from about 1 nm to about 1000 nm, fromabout 10 nm to about 500 nm, from about 25 nm to about 300 nm, fromabout 40 nm to about 250 nm, or from about 50 nm to about 200 nm.

In some embodiments, the microbe-targeting molecules can be modified,e.g., to facilitate attachment of the microbe-targeting molecules to asubstrate. For example, in one embodiment, the microbe-targetingmolecules can be biotinylated, e.g., for attachment to an avidin- oravidin-like coated substrate. Thus, the engineered microbe-targetingmolecules described herein provide a valuable building block for variousapplications, e.g., diagnosis and/or treatment of diseases caused bymicrobes or pathogens, removal of microbes or pathogens from a sample,including bodily fluids and tissues of a subject, foods, water, or anenvironmental surface; and development of targeted drug deliverydevices.

Accordingly, provided herein is directed to an engineeredmicrobe-targeting molecule comprising: (a) at least one microbesurface-binding domain; (b) a substrate-binding domain adapted fororienting the microbe surface-binding domain away from the substrate;and (c) at least one linker between the microbe surface-binding domainand the substrate-binding domain.

In some embodiments, the microbe-surface binding domain can comprise acarbohydrate recognition domain (CRD) or a fragment thereof. In someembodiments, the microbe-surface binding domain can further comprise anon carbohydrate recognition domain or fragment thereof from thecarbohydrate-binding protein, e.g., a neck region of thecarbohydrate-binding protein. As used herein, the term “non carbohydraterecognition domain” refers to the portion or fragment of acarbohydrate-binding protein that does not directly bind with themicrobe surface.

In some embodiments, the CRD or the carbohydrate-binding protein can bederived from, e.g., mannose-binding lectin. Hence, another aspectprovided herein is directed to an engineered mannose-binding lectinmolecule comprising: (a) at least one carbohydrate recognition domain(CRD) or a fragment thereof; (b) a substrate-binding domain adapted fororienting the CRD away from the substrate; and (c) at least one linkerbetween the CRD and the substrate-binding domain.

In some embodiments, the microbe-surface binding domain comprises thefull amino acid sequence of a carbohydrate-binding protein. In someembodiments, the amino acid sequence of the carbohydrate-binding proteindoes not include a complement region. In some embodiments, the aminoacid sequence of the carbohydrate-binding protein does not include acoagulation activation region.

In some embodiments of any aspects described herein, the linker cancomprise a portion of a Fc region of an immunoglobulin, e.g., IgG1. Insuch embodiments, the portion of the Fc region can be linked, directlyor indirectly, to N-terminal of the carbohydrate recognition domain. Insome embodiments, the portion of the Fc region can be geneticallymodified, e.g., to increase half-life of the engineered molecules, ormodulate an immune response (e.g., antibody-dependent cell-mediatedcytotoxicity and complement-dependent cytotoxicity).

In some embodiments of any aspects described herein, thesubstrate-binding domain can comprise at least one oligopeptidecomprising an amino acid sequence of AKT. In other embodiments, thesubstrate-binding domain can comprise a biotin molecule. Depending onvarious applications, e.g., for use as a soluble protein inpharmaceutical compositions, the substrate-binding domain can becomenon-essential in some embodiments of the engineered microbe-targetingmolecules. Otherwise, the engineered microbe-targeting molecules can beused to coat various substrates for a wide variety of applications. Insome embodiments, the substrate is a magnetic microbead, resulting information of a microbe-targeting magnetic microbead or opsonin. In someembodiments, the microbe-targeting magnetic microbead or opsonin canencompass a microbe-targeting nanobead.

Not only can the microbe-targeting magnetic microbeads be used to removemicrobes or pathogens in a sample, e.g., blood and tissues, they canalso be used to develop assays for detecting the presence or absence of,and/or differentiating between, different microbes or pathogens.Accordingly, kits and assays for detecting the presence or absence ofmicrobes, and/or differentiating between, different microbes orpathogens in a test sample are also provided herein. In someembodiments, the kits comprise microbe-targeting substrates (e.g., butnot limited to, one or more containers each containing a population ofmagnetic microbeads coated with a plurality of the engineeredmicrobe-targeting molecules); and at least one reagent. In someembodiments, the kits can further comprise one or more containers eachcontaining a population of detectable labels, wherein each of thedetectable labels is conjugated to a molecule that binds to the microbesor pathogens. Such kits can be used for analysis, e.g., by anenzyme-linked immunosorbent assay (ELISA), fluorescent linkedimmunosorbent assay (FLISA), immunofluorescent microscopy, fluorescencein situ hybridization (FISH), or any other radiological, chemical,enzymatic or optical detection assays. In some embodiments, the kits andassays described herein can be adapted for antibiotic susceptibilitytests, e.g., to determine susceptibility of a microbe in a test sampleto one or more antibiotics, regardless of whether the identity of themicrobe is known or not.

Without limitations, in some embodiments, the engineeredmicrobe-targeting molecules can be formulated as an antibiotic orantiseptic for use in various applications, e.g., wound dressings, aloneor in combination with other wound dressing protocols, e.g., silvernanoparticles and other wound treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C shows a general scheme of engineering one or moreembodiments of engineered microbe-targeting or microbe-binding moleculesand microbe-targeting substrates described herein. FIG. 1A is adiagrammatic view of a native (wild-type) mannose-binding lectin (MBL).FIG. 1B shows one or more embodiments of the engineeredmicrobe-targeting molecules or engineered-binding molecules, e.g.,engineered MBL molecules. FIG. 1C shows one or more embodiments of themicrobe-targeting or microbe-binding molecules conjugated to asubstrate, e.g., a magnetic microbead or nanobead, to form amicrobe-targeting substrate.

FIG. 2 shows a crystal structure of a portion of a wild-type MBL, whichis the “neck and carbohydrate recognition domain (CRD) head.” Thecrystal structure depicts three MBL heads, and calcium binding sites(Chang et al. (1994) J Mol Biol. 241:125-7).

FIG. 3 is a schematic diagram showing an exemplary Fc-X vector constructfor one or more embodiments of the engineered microbe-targeting ormicrobe-binding molecules described herein.

FIG. 4 shows a Western blot image indicating expression of the purifiedwild-type MBL (MBL WT) proteins and one or more embodiments of theengineered microbe-targeting or microbe-binding molecules describedherein (FcMBL.81: SEQ ID NO. 6).

FIG. 5 shows the mannan-binding results of various embodiments of theengineered microbe-targeting or microbe-binding molecules describedherein in the presence or absence of calcium ions. A chelating agent(e.g., EDTA) can be added to the sample to remove calcium ions.

FIGS. 6A and 6B are bar graphs showing the results of capturingmicrobes, e.g., C. albicans, with one or more embodiments of themicrobe-targeting substrates (e.g., AKT-FcMBL.81 conjugated to magneticmicrobeads having a size of about 1 μm at various microbe densities.FIG. 6A shows the percentage of microbes bound to microbe-targetingsubstrates and controls at a low microbe density (e.g., 1500 C. albicanscells). FIG. 6B shows the amount of unbound microbes remained in themicrobe samples after treatment with different magnetic microbeads(including the engineered microbe-targeting magnetic microbeads) whenthe microbe is present at a much higher microbe density (e.g., greaterthan 10⁸ cells).

FIG. 7 shows the size effect of one or more embodiments of themicrobe-targeting substrates (e.g., microbe-targeting magneticmicrobeads such as AKT-FcMBL.81 magnetic microbeads, wherein the size ofthe microbeads were varied from about 100 nm to about 1000 nm diameter)on the efficiency of capturing microbes or pathogens, e.g., Candida.

FIG. 8 shows the amount of unbound microbes remained in the microbesamples after treatment with different magnetic microbeads (includingthe engineered microbe-targeting magnetic microbeads) when the microbes(e.g., Candida) are growing in a log phase vs. in a saturated phase.

FIG. 9 shows the depletion of microbe/microbial matter from a bloodsample from a human donor as measured using FcMBL ELISA. In the figure,“input” corresponds to undiluted EDTA donor blood spiked with S. aureusor E. coli, and supplemented with Ca²⁺ (final [Ca²⁺]=5 mM) and heparin(4 mg/ml), before addition of any FcMBL microbeads. “1^(st) run”corresponds to the “input” blood sample incubated with 20 μl/mL MYONE™FcMBL microbeads for microbe capture (with mixing on a HULAMIXER™ 20′),followed by FcMBL-based ELISA analysis. “2^(nd) run” corresponds to the“input” blood sample incubated with 20 μl/ml MYONE™ FcMBL microbeads formicrobe capture (with mixing on a shaker 10′), followed by FcMBL-basedELISA analysis.

FIG. 10 is a schematic diagram of an exemplary ELISA assay comprisingengineered microbe-targeting magnetic microbeads according to one ormore embodiments. The ELISA assay can be used for any diagnosticapplications, e.g., for sepsis tests.

FIG. 11 is a graph showing results of detecting C. albicans in blood.Serial dilutions of C. albicans were spiked into blood, captured byAKT-FcMBL magnetic microbeads (1 μm) and detected by an ELISA methodusing HRP-labeled FcMBL.

FIG. 12 is a graph showing bacterial detection sensitivity of one ormore embodiments of the FcMBL-based ELISA assay. Serial dilutions of E.coli were spiked into a buffer, captured by AKT-FcMBL magneticmicrobeads (about 128 nm in size) and detected by an ELISA method usingHRP-labeled FcMBL. In some embodiments, the limit of detection (LOD) ofthe FcMBL-based ELISA colorimetric assay is about or below 160 E. colibacteria.

FIG. 13 is a schematic diagram showing one or more embodiments of adipstick assay for microbial detection. The FcMBL can be attached to amembrane (for example Biodyne membrane). The membrane can be mixed witha test sample (e.g., blood sample), washed, incubated with a desireddetecting protein (e.g., AP-labeled FcMBL or specific antibody forcertain microbes, e.g., bacteria or fungus), washed and added with areadout reagent for colorimetric development. The dipstick assay can beperformed manually or modified for automation.

FIG. 14 is a schematic diagram showing one or more embodiments of anELISA-based test for microbial detection. A test sample (e.g., bloodsample) can be added into a single tube (e.g., a blood collectioncontainer such as EDTA VACUTAINER®) containing lyophilized FcMBLmagnetic microbeads or FcMBL-coated magnetic microbeads. An exemplaryprotocol for microbial capture and detection is described in Example 10.The ELISA-based test can be performed manually or modified forautomation. In some embodiments, the single-tube based ELISA assay canbe used to detect microbes or pathogens such as S. aureus and E. coli.

FIG. 15 is an image showing direct detection of bacteria on a membraneby AP-labeled FcMBL. Serial dilutions of E. coli and S. aureus (10⁻¹ to10⁻⁶) were spotted directly onto a Biodyne membrane, blocked for about30 mins in 1% casein, washed twice in TBST containing Ca²⁺ (5 mM),incubated with AP-labeled FcMBL (1:10,000 dilution) in 3% BSA 1×TBSTcontaining Ca²⁺ (for about 20 min), washed twice in TBST containing Ca²⁺(5 mM) and once in TBS containing Ca²⁺ (5 mM), and reacted with BCIP/NBTfor about 20 mins to develop a colorimetric readout. In this example,maximum dilution allowed for detection of both species was 10⁻⁴ after 30min development (corresponding to detection of 130 E. coli and 343 S.aureus cells).

FIG. 16 is an image showing capture and detection of S. aureus by dotblot using a membrane coupled with FcMBL. Dilutions of S. aureus (10⁻²and 10⁻⁴) were captured by FcMBL immobilized on a Biodyne membrane. Forexample, 5 μL of two indicated concentrations of FcMBL were spotted ontoa Biodyne membrane, allowed to dry, blocked in 1% casein, and washedtwice in TBST containing Ca²⁺ (5 mM). Each FcMBL concentration wasassessed for capture (˜10 min) of serial dilutions of S. aureus, washed,and detected with 1:10,000 dilution of AP-labeled FcMBL in 3% BSA 1×TBSTcontaining Ca²⁺ (˜20 min). Excess AP-labeled FcMBL was removed by washes(e.g., washing three times with TBST containing Ca²⁺ (5 mM) and oncewith TBS containing Ca²⁺ (5 mM)). Colorimetric detection was developedwith BCIP/NBT for ˜20 min.

FIG. 17 is a schematic of an exemplary microbial detection process ordiagnosis process.

FIGS. 18A and 18B are line graphs showing ELISA of E. coli on twodifferent FcMBL microbead formats. FIG. 18A corresponds to FcMBLdirectly coupled to MYONE™ Tosyl activated beads and FIG. 18Bcorresponds to biotinylated AKT-FcMBL coupled to Streptavidin MYONE™ T1microbeads (˜1000 nm diameter). Three different dilutions of an E. coliovernight culture were captured on FcMBL microbeads, washed with one offour elution buffers and then run through one or more embodiments of theELISA protocol described herein. A decrease in signal corresponds tofewer E. coli bound to the microbeads prior to the ELISA detection.

FIG. 19 is an image showing plating out of equal titers of S. aureuseither mixed with FcMBL microbeads or control without FcMBL microbeads.

FIGS. 20A and 20B are line graphs showing capture efficiency ofengineered microbe-targeting or microbe-binding molecules (e.g., FcMBL)in clinical isolates of different microbial species. FIG. 20A shows datafor capture efficiency of FcMBL in the clinical isolates of S. aureusand methicillin-resistant S. aureus (MRSA). FIG. 20B shows data forcapture efficiency of FcMBL in the clinical isolates of S. aureus, MRSA,N. meningitidis, and P. aeroginosa.

FIGS. 21A-21B are line graphs showing capture efficiency of engineeredmicrobe-targeting or microbe-binding molecules (e.g., FcMBL) in clinicalisolates obtained from different types of fluids. FIGS. 21A and 21Bshows data for capture efficiency of FcMBL in the clinical isolates ofS. aureus and E. coli, respectively, obtained from other body fluids,e.g., urine, cerebrospinal fluid (CSF), and sputum.

FIG. 22 is a schematic diagram showing mechanism by which S. aureusavoids opsonophagocytosis. See additional details in Fraser T., NatureReviews Microbiology 2005: 3(12):948-58.

FIG. 23 is a bar graph showing detection signals of variousconcentrations of S. aureus captured by AKT-FcMBL 1 μM magneticmicrobeads and detected by FcMBL-HRP ELISA. Sensitivity of thisembodiment of the assay was about 149 CFU/mL.

FIG. 24 is a bar graph showing elution of S. aureus and E. coli bacteriabound onto FcMBL-coated substrates (e.g., magnetic microbeads) withdifferent treatments, including chelation, pH and salt washes.

FIGS. 25A and 25B are bar graphs showing elution of E. coli and S.aureus off FcMBL-coated substrates (e.g., magnetic microbeads) usingchelators. FIG. 25A shows the results in OD450 and FIG. 25B shows theresults as a percent of bound bacteria remained on the FcMBL-coatedsubstrates after treatment.

FIGS. 26A and 26B show results of tube-based ELISA for S. aureus and E.coli binding to FcMBL-coated substrates (e.g., magnetic microbeads) inthe presence of a chelating agent (e.g., EDTA). FIG. 26A is an imageshowing colorimetric outcomes of the tube-based ELISA for S. aureus andE. coli binding to FcMBL-coated substrates (e.g., magnetic microbeads)in the presence or absence of a chelating agent (e.g., EDTA). FIG. 26Bis a bar graph showing quantitative measurement of the color developedin FIG. 26A.

FIG. 27 is a bar graph comparing different microbial or pathogenicspecies captured on FcMBL-coated substrates (e.g., magnetic microbeads)in the presence or absence of a chelating agent (e.g., EDTA) and variousCa²⁺ concentrations.

FIG. 28 is an image showing colorimetric outcomes of the tube-basedELISA assay for S. aureus and E. coli binding to FcMBL-coated substrates(e.g., magnetic microbeads) in the presence or absence of a chelatingagent (e.g., EDTA) and/or a low pH buffer.

FIG. 29 is an image showing dot blot determination of E. coli and S.aureus with or without EDTA in the capture and/or wash buffer.

FIGS. 30A-30B are images showing binding of one or more embodiments ofmicrobe-targeting substrates to microbial matter, including livemicrobes and/or fragments or matter derived from microbes. FIG. 30Ashows that microbial outgrowth is observed when one or moremicrobe-targeting substrates (e.g., FcMBL-coated fluorescent microbeads)bind(s) to at least one live microbe, e.g., E. coli. FIG. 30B is a setof fluorescent images showing that FcMBL-coated fluorescent microbeadsbind to microbial matter (left panel) including live microbes (indicatedby the middle panel) and fragments or matter derived from microbes. Theright panel is an overlay of the first two fluorescent images inaddition to a bright-field image.

FIGS. 31A-31B are images showing capture of microbes or fragmentsthereof on one or more embodiments of microbe-targeting substrates fromfluid samples, followed by antibody characterization. FIG. 31A showscapture of E. coli or fragments thereof on FcMBL-coated microbeads(e.g., magnetic or fluorescent microbeads) from heparinized blood,followed by incubation with an antibody against E. colilipopolysaccharide lipid A (anti-LPS lipid A antibody. FIG. 31B showscapture of E. coli or fragments thereof on FcMBL-coated microbeads(e.g., magnetic or fluorescent microbeads) from blood containing EDTAanticoagulation agent, followed by incubation with an antibody againstE. coli lipopolysaccharide lipid A (anti-LPS lipid A antibody). BothFIGS. 31A-31B show that the anti-LPS lipid A antibody does not bind toFcMBL-coated microbeads in the absence of E. coli or fragments thereof.

FIGS. 32A-32B are images showing capture of microbes on one or moreembodiments of microbe-targeting substrates from samples of a rat sepsismodel, followed by antibody characterization. FIG. 32A shows capture ofmicrobes or fragments thereof on FcMBL-coated microbeads (e.g., magneticor fluorescent microbeads) from rat blood (upper panel) or pleural(lower panel) fluids after 24-hr infection, followed by incubation withan anti-LPS lipid A antibody. FIG. 32B shows capture of microbes orfragments thereof on FcMBL-coated microbeads (e.g., magnetic orfluorescent microbeads) from rat blood (upper panel) or pleural (lowerpanel) fluids after 72-hr infection, followed by incubation with ananti-LPS lipid A antibody.

FIG. 33 is a set of images showing the use of specific antibodies tomicrobes to allow further discrimination or identification of samplesthat indicate positive signals with one or more embodiments ofmicrobe-targeting substrates. De-identified clinical blood samples werescreened by FcMBL ELISA described herein and the captured microbialmatters (including intact cells and fragments thereof) on theFcMBL-coated microbeads were further screened by using an anti-LPS lipidA antibody. The top panel indicates that no detection of anti-LPS lipidA antibody signal was observed in clinical samples with substantiallynegative or negligible signal from FcMBL ELISA, indicative of nomicrobial infection detected in the clinical samples. The middle panelindicates that the microbial matter producing positive signal (OD=˜1.69)in FcMBL ELISA bound to anti-LPS lipid A antibody, which indicates thatthe microbial matter could be derived from E. coli, and that thecorresponding clinical samples had a gram-negative infection (e.g., E.coli infection). In contrast, the bottom panel indicates that themicrobial matter producing positive signal (OD>3.9) did not bind toanti-LPS lipid A antibody, which indicates that the microbial mattercould be derived from microbes other than E. coli, e.g., when theclinical samples were infected with a gram-positive microbe.

FIGS. 34A-34D are data graphs showing that use of FcMBL magneticmicrobeads is a more sensitive and reliable measure of blood-bornepathogens (including live and non-viable pathogens such as deadpathogens and endotoxins) than conventional blood cultures. FIG. 34A isa bar graph showing results of anaerobe cultures at Day 4 of bloodcollected from five rats developed with intra-abdominal abscesses. FIG.34B is a plot comparing the microbe detection results based oncolorimetric ELISA using FcMBL magnetic microbeads and conventionalblood cultures and their correlations with morbidity of the rats. FIG.34C is a line graph showing correlation of pathogen load determined bythe ELISA using FcMBL magnetic microbeads with morbidity ranking. FIG.34D is a bar graph comparing the microbe detection results based oncolorimetric ELISA using FcMBL magnetic microbeads and conventionalblood cultures in a separate experiment.

FIG. 35 is a bar graph showing percentages of microbe depletion by oneor more embodiments of the microbe-targeting magnetic microbeads.FcMBL-coated magnetic microbeads of different sizes (˜1 μm, ˜128 nm, and˜50 nm) were used to capture E. coli and S. aureus that were initiallyspiked into a buffered solution. The microbe-bound FcMBL-coated magneticmicrobeads were then removed from the buffered solution. After removalof the magnetic microbeads, the buffered solution was used forinoculation on LB plates to determine the level of microbe depletion byFcMBL-coated magnetic microbeads of different sizes.

DETAILED DESCRIPTION OF THE INVENTION

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such may vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention, which is defined solely by the claims.

As used herein and in the claims, the singular forms include the pluralreference and vice versa unless the context clearly indicates otherwise.Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.”

All patents and other publications identified are expressly incorporatedherein by reference for the purpose of describing and disclosing, forexample, the methodologies described in such publications that might beused in connection with the present invention. These publications areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing in this regard should be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention or for any other reason. Allstatements as to the date or representation as to the contents of thesedocuments is based on the information available to the applicants anddoes not constitute any admission as to the correctness of the dates orcontents of these documents.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as those commonly understood to one of ordinaryskill in the art to which this invention pertains. Although any knownmethods, devices, and materials may be used in the practice or testingof the invention, the methods, devices, and materials in this regard aredescribed herein.

Described herein are engineered microbe-targeting or microbe-bindingmolecules, compositions comprising the same, processes or assays, andkits for separating microbes from a test sample in vivo, in situ or invitro, and/or detecting the presence or absence of the microbes in thetest sample. The engineered microbe-targeting or microbe-bindingmolecules can bind or capture at least one microbe, e.g., an intactmicrobe, and/or “microbial matter.” The term “microbial matter” as usedherein refers to any matter or component that is derived, originated orsecreted from a microbe. For example, microbial matter or a componentderived or secreted from a microbe that can bind to an engineeredmicrobe-targeting or microbe-binding molecule can include, but are notlimited to, a cell wall component, an outer membrane, a plasma membrane,a ribosome, a microbial capsule, a pili or flagella, any fragments ofthe aforementioned microbial components, any nucleic acid (e.g., DNA,including 16S ribosomal DNA, and RNA) derived from a microbe, andmicrobial endotoxin (e.g., lipopolysaccharide). In addition, microbialmatter can encompass non-viable microbial matter that can cause anadverse effect (e.g., toxicity) to a host or an environment.

In accordance with various embodiments described herein, the engineeredmicrobe-targeting molecules or microbe-binding molecules comprise amicrobe surface-binding domain (e.g., a carbohydrate recognitiondomain), directly or indirectly, conjugated to a linker (e.g., a Fcfragment), which can further comprise a substrate-binding domain forimmobilization. Thus, the engineered microbe-targeting molecules ormicrobe-binding molecules described herein can be used as solubleproteins, e.g., in therapeutic compositions, or be immobilized to asubstrate for various applications ranging from diagnosis and/ortreatment of a microbial infection or disease, to microbe-clearingcompositions or devices, to drug delivery.

In one aspect, provided herein is an engineered microbe-targetingmolecule (or an engineered microbe-binding molecule) comprising at leastone microbe surface-binding domain, a substrate-binding domain adaptedfor orienting the carbohydrate recognition domain away from thesubstrate, and at least one linker between the microbe surface-bindingdomain and the substrate-binding domain. In some embodiments, themicrobe surface-binding domain can comprise a carbohydrate recognitiondomain or a fragment thereof. In some embodiments, the microbesurface-binding domain can further comprise at least a portion ofmannose-binding lectin (MBL). Accordingly, another aspect providedherein is an engineered MBL molecule comprising at least a fragment of acarbohydrate recognition domain derived from MBL; a substrate-bindingdomain adapted for orienting the carbohydrate domain away from thesubstrate; and at least one linker between the fragment of the MBLcarbohydrate recognition domain and the substrate-binding domain. Theterms “microbe-binding molecule(s)” and “microbe-targeting molecule(s)”are used interchangeably herein.

In some embodiments of any aspects described herein, thesubstrate-binding domain adapted for orienting the carbohydraterecognition domain away from the substrate is not always necessary andthus can be excluded under certain circumstances, e.g., using theengineered microbe-targeting molecules in a soluble format, e.g., fortherapeutic purposes. Further, it should be noted that the engineeredmicrobe-binding molecules excluding the substrate-binding domain adaptedfor orienting the carbohydrate recognition domain away from thesubstrate does not necessarily mean that the engineered microbe-bindingmolecules cannot bind to a substrate surface. In some embodiments, theengineered microbe-binding molecules excluding the substrate-bindingdomain adapted for orienting the carbohydrate recognition domain awayfrom the substrate can still bind to a substrate surface, but theorientation of the carbohydrate recognition domain relative to thesubstrate surface can be random.

In some embodiments of any aspects described herein, the engineeredmicrobe-targeting molecule can further comprise a detectable label,e.g., to facilitate detection of the presence or absence of a microbeand/or microbial matter. Detectable labels suitable for conjugation tosome embodiments of the engineered microbe-targeting molecule caninclude any composition detectable by spectroscopic, photochemical,biochemical, immunochemical, electrical, magnetic, optical or chemicalmeans, as well as any examples of detectable labels described herein andany equivalent thereof. In some embodiments, the detectable labels alsoencompass any imaging agent (e.g., but not limited to, a bubble, aliposome, a sphere, a contrast agent, or any detectable label describedherein) that can facilitate imaging or visualization of a tissue or anorgan in a subject, e.g., for diagnosis of an infection.

In some embodiments, the detectable label conjugated to the engineeredmicrobe-targeting molecule can include an enzyme of horseradishperoxidase (HRP), alkaline phosphatase (AP), or any combinationsthereof. Conjugation of the detectable label (e.g., HRP or AP) to anyproteins and antibodies are known in the art. In one embodiment,FcMBL-HRP or FcMBL-AP construct is generated using any art-recognizedmethods for direct coupling HRP or AP to FcMBL.

In some embodiments, the detectable label conjugated to the engineeredmicrobe-targeting molecule can include a microbial enzyme substrateconjugated to a detectable agent. For example, the detectable agent canbe any moiety that, when cleaved from a microbial enzyme substrate bythe enzyme possessed or secreted by the microbe, forms a detectablemoiety (e.g., a light-emitting signal), but that is not detectable inits conjugated state. The microbial enzyme substrate is a substratespecific for one or more types of microbes to be detected, and it can beselected depending upon what enzymes the microbe possesses or secretes.See, e.g., International Patent Application: WO 2011/103144 for the useof such detectable label in detection of microbes, the content of whichis incorporated herein by reference.

General methods of preparing any embodiments of the engineeredmicrobe-targeting molecules are known in the art (Ashkenazi, A. and S.M. Chamow (1997), “Immunoadhesins as research tools and therapeuticagents,” Curr. Opin. Immunol. 9(2): 195-200, Chamow, S. M. and A.Ashkenazi (1996). “Immunoadhesins: principles and applications,” TrendsBiotechnol. 14(2):52-60). In one example, an engineeredmicrobe-targeting molecule can be made by cloning into an expressionvector such as Fc-X vector as discussed in Lo et al. (1998) 11:495 andExample 1.

The engineered microbe-targeting molecules can contain sequences fromthe same species or from different species. For example, an interspecieshybrid microbe-targeting molecule can contain a linker, e.g., a peptidelinker, from a murine species, and a human sequence from a carbohydraterecognition domain protein, provided that they do not provideunacceptable levels of deleterious effects. The engineeredmicrobe-targeting molecules described herein can also include those thatare made entirely from murine-derived sequences or fully human.

Microbe Surface-Binding Domain and Carbohydrate Recognition Domain

As disclosed herein, an engineered microbe-targeting molecule cancomprise at least one microbe surface-binding domain, including at leasttwo, at least three, at least four, at least five, at least six, atleast seven, at least eight, at least nine, at least ten or more microbesurface-binding domains. The term “microbe surface-binding domain” asused herein refers to any molecule or a fragment thereof that canspecifically bind to the surface of a microbe or pathogen, e.g., anycomponent present on a surface of a microbe or pathogen, and/or anymicrobial matter, e.g., any matter or component/fragment that isderived, originated or secreted from a microbe. Molecules that can beused in the microbe surface-binding domain can include, for example, butare not limited to, peptides, polypeptides, proteins, peptidomimetics,antibodies, antibody fragments (e.g., antigen binding fragments ofantibodies), carbohydrate-binding protein, e.g., a lectin,glycoproteins, glycoprotein-binding molecules, amino acids,carbohydrates (including mono-, di-, tri- and poly-saccharides), lipids,steroids, hormones, lipid-binding molecules, cofactors, nucleosides,nucleotides, nucleic acids (e.g., DNA or RNA, analogues and derivativesof nucleic acids, or aptamers), peptidoglycan, lipopolysaccharide, smallmolecules, and any combinations thereof. In some embodiments, themicrobe surface-binding domain can comprise a carbohydrate recognitiondomain or a fragment thereof. In some embodiments, a microbesurface-binding domain can comprise a peptidomimetic that mimics anymolecule or a fragment thereof that can specifically bind to the surfaceof a microbe or pathogen, and/or any microbial matter. For example, amicrobe surface-binding domain can comprise a peptidomimetic that mimicsany carbohydrate recognition domain or a fragment thereof, e.g.,carbohydrate recognition domain of MBL or a fragment thereof; or anycarbohydrate recognition domain that is known in the art or a fragmentthereof. In some embodiments, the microbe-surface binding domaincomprises the full amino acid sequence of a carbohydrate-bindingprotein.

In some embodiments, the microbe surface-binding domain can have anamino acid sequence of about 10 to about 300 amino acid residues, orabout 50 to about 150 amino acid residues. In some embodiments, themicrobe surface-binding domain can have an amino acid sequence of atleast about 5, at least about 10, at least about 15, at least about 20,at least about 30, at least about 40, at least about 50, at least about60, at least about 70, at least about 80, at least about 90, at leastabout 100 amino acid residues or more. For any known sequences ofmicrobe surface-binding molecules, one of skill in the art can determinethe optimum length of amino acid sequence for the microbesurface-binding domain.

In some embodiments, the microbe surface-binding domain can comprise anopsonin or a fragment thereof. The term “opsonin” as used herein refersto naturally-occurring and synthetic molecules which are capable ofbinding to or attaching to the surface of a microbe or a pathogen, ofacting as binding enhancers for a process of phagocytosis. Examples ofopsonins which can be used in the engineered molecules described hereininclude, but are not limited to, vitronectin, fibronectin, complementcomponents such as Clq (including any of its component polypeptidechains A, B and C), complement fragments such as C3d, C3b and C4b,mannose-binding protein, conglutinin, surfactant proteins A and D,C-reactive protein (CRP), alpha2-macroglobulin, and immunoglobulins, forexample, the Fc portion of an immunoglobulin.

In some embodiments, the microbe surface-binding domain can comprise acarbohydrate recognition domain. In some embodiments, the microbesurface-binding domain can further comprise at least a portion of acarbohydrate-binding protein or a portion thereof. In some embodiments,the portion of the carbohydrate-binding proteins can activate thecomplement system. In alternative embodiments, the portion of thecarbohydrate-binding protein cannot activate the complement system. Insome embodiments, the portion of the carbohydrate-binding protein can beselected or configured such that it cannot activate the complementsystem, e.g., via modification. Examples of carbohydrate-bindingproteins include, but are not limited to, lectin, collectin, ficolin,mannose-binding lectin (MBL), maltose-binding protein, arabinose-bindingprotein, and glucose-binding protein. Additional carbohydrate-bindingproteins that can be included in the microbe surface-binding domaindescribed herein can include, but is not limited to, lectins oragglutinins that are derived from a plant, e.g., Galanthus nivalisagglutinin (GNA) from the Galanthus (snowdrop) plant, and peanut lectin.In some embodiments, pentraxin family members, e.g., C-reactive protein,can also be used as a carbohydrate-binding protein. Pentraxin familymembers can generally bind capsulated microbes. The carbohydrate-bindingproteins can be wild-type, recombinant or a fusion protein. Therespective carbohydrate recognition domains for suchcarbohydrate-binding proteins are known in the art, and can be modifiedfor various embodiments of the engineered microbe-targeting moleculesdescribed herein. In some embodiments, peptidomimetics or any structuralmimics mimicking a microbe surface-binding domain (e.g., a carbohydraterecognition domain or a fragment thereof) and capable of binding to amicrobe surface can also be used as a microbe surface-binding domaindescribed herein.

The term “lectin” as used herein refers to any molecules includingproteins, natural or genetically modified (e.g., recombinant), thatinteract specifically with saccharides (e.g., carbohydrates). The term“lectin” as used herein can also refer to lectins derived from anyspecies, including, but not limited to, plants, animals, insects andmicroorganisms, having a desired carbohydrate binding specificity.Examples of plant lectins include, but are not limited to, theLeguminosae lectin family, such as ConA, soybean agglutinin, peanutlectin, lentil lectin, and Galanthus nivalis agglutinin (GNA) from theGalanthus (snowdrop) plant. Other examples of plant lectins are theGramineae and Solanaceae families of lectins. Examples of animal lectinsinclude, but are not limited to, any known lectin of the major groupsS-type lectins, C-type lectins, P-type lectins, and I-type lectins, andgalectins. In some embodiments, the carbohydrate recognition domain canbe derived from a C-type lectin, or a fragment thereof. C-type lectincan include any carbohydrate-binding protein that requires calcium forbinding. In some embodiments, the C-type lectin can include, but are notlimited to, collectin, DC-SIGN, and fragments thereof. Without wishingto be bound by theory, DC-SIGN can generally bind various microbes byrecognizing high-mannose-containing glycoproteins on their envelopesand/or function as a receptor for several viruses such as HIV andHepatitis C.

Collectins are soluble pattern recognition receptors (PRRs) belonging tothe superfamily of collagen containing C-type lectins. Exemplarycollectins include, without limitations, mannose-binding lectin (MBL)(also known as mannan-binding lectin, mannan-binding protein, ormannose-binding protein), surfactant protein A (SP-A), surfactantprotein D (SP-D), collectin liver 1 (CL-L1), collectin placenta 1(CL-P1), conglutinin, collectin of 43 kDa (CL-43), collectin of 46 kDa(CL-46), and a fragment thereof.

Mannose-binding lectin (MBL), also known as mannose binding protein(MBP), or mannan-binding lectin or mannan-binding protein, is acalcium-dependent serum protein that can play a role in the innateimmune response by binding to carbohydrates on the surface of a widerange of microbes or pathogens (viruses, bacteria, fungi, protozoa)where it can activate the complement system. MBL can also serve as adirect opsonin and mediate binding and uptake of pathogens by taggingthe surface of a pathogen to facilitate recognition and ingestion byphagocytes.

MBL is a member of the collectin family of proteins. A native MBL is amultimeric structure (e.g., about 650 kDa) composed of subunits, each ofwhich contains three identical polypeptide chains (FIG. 1A). Each MBLpolypeptide chain (containing 248 amino acid residues in length with asignal sequence: SEQ ID NO. 1) comprises a N-terminal cysteine richregion, a collagen-like region, a neck region, and a carbohydraterecognition domain (CRD). The sequence of each region has beenidentified and is well known in the art. SEQ ID NO. 2 shows afull-length amino acid sequence of MBL without a signal sequence.

The surface or carbohydrate recognition function of a native MBL ismediated by clusters of three C-type carbohydrate-recognition domains(CRDs) held together by coiled-coils of a-helices. The N-terminalportion collagen-like domain is composed of Gly-X-Y triplets. The shortN-terminal domain contains several cysteine residues that forminterchain disulfide bonds. Serum MBLs assemble into larger formscontaining 2-4 trimeric subunits in rodents and as many as six subunitsin humans. All three oligomeric forms of rat serum MBP, designated MBPA,can fix complement, although the larger oligomers have higher specificactivity. Many species express a second form of MBP. In rats, the secondform, MBP-C, is found in the liver. MBP-C does not form higher oligomersbeyond the simple subunit that contains three polypeptides.

When a native MBL interacts with carbohydrates on the surface ofmicrobes or pathogens, e.g., calcium-dependent binding to thecarbohydrates mannose, N-acetylglucosamine, and/or fucose, it can formthe pathogen recognition component of the lectin pathway of complementactivation. The MBL binds to surface arrays containing repeated mannoseor N-acetylglucosamine residues. It circulates as a complex with one ormore MBP-associated serine proteases (MASPs) that autoactivate when thecomplex binds to an appropriate surface. The MBL and associated MASPproteins can activate C2/C4 convertase leading to the deposition of C4on the pathogen surface and opsonization for phagocytosis. The nativeMBL can also activate coagulation function through MASP proteins.

While native MBL can detect microbes or pathogens and act as opsoninsfor tagging the microbes for phagocytosis, native MBLs may not bedesirable for use in treatment of microbe-induced inflammatory diseasesor infections, e.g., sepsis, because native MBLs can activate complementsystem and induce an inflammatory response. Provided herein is anengineered MBL molecule that binds to microbes or pathogens, comprisingat least one carbohydrate recognition domain or a fragment thereof,e.g., derived from MBL. In some embodiments, the engineered MBL moleculecan comprises at least two, at least three or at least four carbohydraterecognition domains or a fragment thereof. In some embodiments, theengineered MBL molecules do not activate complement system orcoagulation side effects that are present in a native MBL. Suchembodiments can be used as dominant-negative inhibitors of downstreamresponses in vivo or as microbe-binding proteins that do not inducecoagulation or complement fixation in vitro. For example, the engineeredMBL molecules that do not have complement fixation and/or coagulationdomains can act as a dominant negative protein in terms of activatingcytokine and/or inflammatory cascades, and thus reduce systeminflammatory syndrome and/or sepsis symptoms.

FIG. 1B shows a diagrammatic view of a dimeric engineered MBL molecule100 according to one or more embodiments of the engineered MBL moleculesdescribed herein. The dimeric molecule 100 comprises at least twocarbohydrate recognition domains 102 (e.g., MBL CRD) connected, directlyor indirectly, to a linker, e.g., a Fc region 106. The N-terminal of theFc region 106 can further comprise an oligopeptide 108, e.g., comprisingan amino acid sequence AKT. In some embodiments, the carbohydraterecognition domains 102 can further comprise neck regions 104 such asMBL neck to provide flexibility of the CRD interacting with microbes.

The full-length amino acid sequence of carbohydrate recognition domain(CRD) of MBL is shown in SEQ ID NO. 4. The carbohydrate recognitiondomain of an engineered MBL described herein can have an amino acidsequence of about 10 to about 300 amino acid residues, or about 50 toabout 160 amino acid residues. In some embodiments, the microbesurface-binding domain can have an amino acid sequence of at least about5, at least about 10, at least about 15, at least about 20, at leastabout 30, at least about 40, at least about 50, at least about 60, atleast about 70, at least about 80, at least about 90, at least about100, at least about 150 amino acid residues or more. Accordingly, insome embodiments, the carbohydrate recognition domain of the engineeredMBL molecule can comprise SEQ ID NO. 4. In some embodiments, thecarbohydrate recognition domain of the engineered MBL molecule cancomprise a fragment of SEQ ID NO. 4. Exemplary amino acid sequences ofsuch fragments include, but are not limited to, ND (SEQ ID NO. 10), EZN(SEQ ID NO. 11: where Z is any amino acid, e.g., P), NEGEPNNAGS (SEQ IDNO. 12) or a fragment thereof comprising EPN, GSDEDCVLL (SEQ ID NO. 13)or a fragment thereof comprising E, and LLLKNGQWNDVPCST (SEQ ID NO. 14)or a fragment thereof comprising ND. Modifications to such CRDfragments, e.g., by conservative substitution, are also within the scopedescribed herein. In some embodiments, the MBL or a fragment thereofused in the microbe surface-binding domain of the engineeredmicrobe-targeting molecules described herein can be a wild-type moleculeor a recombinant molecule.

The exemplary sequences provided herein for the carbohydrate recognitiondomain of the engineered microbe-targeting molecules are not construedto be limiting. For example, while the exemplary sequences providedherein are derived from a human species, amino acid sequences of thesame carbohydrate recognition domain in other species such as mice,rats, porcine, bovine, feline, and canine are known in the art andwithin the scope described herein.

In some embodiments, the nucleic acid encodes a carbohydrate recognitiondomain having greater than 50% homology, including greater than 60%,greater than 70%, greater than 80%, greater than 90% homology or higher,to a fragment of at least 50, at least 60, at least 70, at least 80, atleast 90, at least 100, at least 150 contiguous amino acids or more, ofany known carbohydrate-binding molecules (e.g., mannose-bindinglectins).

The term “carbohydrate recognition domain” as used herein refers to aregion, at least a portion of which, can bind to carbohydrates on asurface of microbes or pathogens. For example, as shown in FIG. 1B, thecarbohydrate recognition domain, in some embodiments, can encompass MBLCRD 102. However, in some embodiments, the carbohydrate recognitiondomain can be also construed to encompass a neck region 104 in additionto MBL CRD 102. In some embodiments, the carbohydrate recognition domaincan comprise at least about 50% of its domain, including at least about60%, at least about 70%, at least about 80%, at least about 90% orhigher, capable of binding to carbohydrates on a microbe surface. Insome embodiments, 100% of the carbohydrate recognition domain can beused to bind to microbes or pathogens. In other embodiments, thecarbohydrate recognition domain can comprise additional regions that arenot capable of carbohydrate binding, but can have other characteristicsor perform other functions, e.g., to provide flexibility to thecarbohydrate recognition domain when interacting with microbes orpathogens.

Accordingly, in some embodiments, the carbohydrate recognition domaincan further comprise a neck region of the MBL with an amino acidsequence pdgdsslaaserkalqtema rikkwltfslgkq (SEQ ID NO. 15) or afragment thereof. Without wishing to be bound by theory, the neck regioncan provide flexibility and proper orientation of the CRD to bind to amicrobe surface. In some embodiments, the carbohydrate recognitiondomain can comprises a full-length CRD of MBL (SEQ ID NO. 4; termed as“CRD head” 102) and the neck region thereof 104, as shown in FIG. 1B.The amino acid sequence encoding a full-length CRD of MBL and the neckregion thereof is shown in SEQ ID NO. 5. The crystal structure of anative MBL “neck and CRD head” has been previously shown in Chang et al.(1994) J Mol Biol. 241:125-7 (FIG. 2). A skill artisan can readilymodify the identified CRD and fragments thereof to modulate itsorientation and binding performance to carbohydrates on a microbesurface, e.g., by theoretical modeling and/or in vitrocarbohydrate-binding experiments. In addition, based on the crystalstructure of the native MBL “neck and CRD head”, peptidomimetics thatcan effectively mimic at least a fragment of the CRD head and optionallythe neck region can be also used as a carbohydrate recognition domain ofthe engineered microbe-targeting molecule or MBL molecule describedherein. One of skill in the art can readily determine suchpeptidomimetic structure without undue experimentations, using anymethods known in the art and the known crystal structure.

In some embodiments, the carbohydrate recognition domain of themicrobe-targeting molecule can further comprise a portion of acarbohydrate-binding protein. However, in some circumstances, complementor coagulation activation induced by a carbohydrate-binding protein or afragment thereof can be undesirable depending on various applications,e.g., in vivo administration for treatment of sepsis. In suchembodiments, the portion of the carbohydrate-binding protein can excludeat least one of complement and coagulation activation regions. By way ofexample, when the carbohydrate-binding protein is mannose-binding lectinor a fragment thereof, the mannose-binding lectin or a fragment thereofcan exclude at least one of the complement and coagulation activationregions located on the collagen-like region. In such embodiments, themannose-binding lectin or a fragment thereof can exclude at least aboutone amino acid residue, including at least about two amino acidresidues, at least about three amino acid residues, at least about fouramino acid residues, at least about five amino acid residues, at leastabout six amino acid residues, at least about seven amino acid residues,at least about eight amino acid residues, at least about nine amino acidresidues, at least about ten amino acid residues or more, around aminoacid residue K55 or L56 of SEQ ID NO. 2. Exemplary amino sequencescomprising K55 or L56 of SEQ ID NO. 2 that can be excluded from theengineered MBL molecule include, but are not limited to,EPGQGLRGLQGPPGKLGPPGNPGPSGS (SEQ ID NO. 16), GKLG (SEQ ID NO. 17),GPPGKLGPPGN (SEQ ID NO. 18), RGLQGPPGKL (SEQ ID NO. 19), GKLGPPGNPGPSGS(SEQ ID NO. 20), GLRGLQGPPGKLGPPGNPGP (SEQ ID NO. 21), or any fragmentsthereof.

Further regarding the carbohydrate recognition domain (CRD) or afragment thereof, its binding characteristics can be manipulated bydirected evolution for altered binding specificity. By way of exampleonly, MBL can be modified so that it binds to a more limited set ofsugars or other molecular features, with the result that the modifiedMBL will bind to a more limited set of microbes to provide a capabilityfor pathogen class identification (e.g., one of virus, bacteria, fungi,or protozoan), subclass typing (e.g., gram negative or gram positivebacteria) or specific species determination. Numerous strategies ofdirected evolution are available in the art.

For example, a straightforward directed evolution strategy visuallyexamines an atomic structure of MBL complexed with a sugar, and thenmutates appropriate amino acids that make contact in a sugar-specificmanner, so that distinctive contacts are lost or particular types ofsteric hindrance are created. The three dimensional structure of rat MBLhas been solved in a complex with a high-mannose oligosaccharide andwith N acetylglucosamine, a methylated fucose, and so on. His189Val andIle207Val are examples of substitutions that modifications alterspecificity.

In another strategy of directed evolution, the protein is subjected torandom mutagenesis and the resulting proteins are screened for desiredqualities. This is a particularly useful technology for affinitymaturation of phage display antibodies, where the antibody complementarydetermining regions (CDRs) are mutated by saturation mutagenesis andsuccessful variants of the six CDRs are shuffled together to form thehighest affinity antibodies.

The directed evolution paradigm can be applied to MBL in order to selectMBL variants with specific binding to, e.g., but not limited to, yeast,gram-positive bacteria, gram-negative, coagulase negative, and aerobicbacteria. For this to work, however, the pattern and nature of thetarget sugars or related surface features on these target microorganismscan differ between the classes or species.

MBL is known to bind strongly to mannose and N-acetylglucosamine sugarson fungi, gram-positive, and gram-negative bacteria. For example, MBLbinds strongly to Candida spp., Aspergillus fumigatus, Staphylococcusaureus, and β hemolytic group A streptococci. MBL has intermediateaffinity to Escherichia coli, Klebsiella spp., and Haemophilusinfluenzae type b. MBL binds weakly to β hemolytic group B streptococci,Streptococcus pneumoniae, and Staphylococcus epidermidis. Neth et al.,68 Infect. & Immun. 688 (2000). The capsular polysaccharide of Neisseriameningitides serogroup B, H. influenzae type b and Cryptococcusneoformans are thought to decrease MBL binding, as does bacterialendotoxin. Id.; Van Emmerik et al., 97 Clin. Exp. Immunol. 411 (1994);Schelenz et al., 63 Infect. Immun. 3360 (1995).

Others have reported that MBL facilitates opsonophagocytosis of yeastsbut not of bacteria, despite MBL binding: MBL (Lectin) pathway ofcomplement was critical for the opsonophagocytosis of yeast, but theclassical complement pathway was critical for opsonophagocytosis ofbacteria. Brouwer et al., 180 J. Immunol. 4124 (2008). It was notreported that MBL bound to the bacterial species tested, however, onlythat MBL binding did not promote significant complement activation andopsonophagocytosis.

Derivatives of MBL with a particular specificity can be isolated, e.g.,by the following approach, which is a standard phage display strategy:First, express a set of MBL variants from a phagemid vector; then bindthis library to a target of interest (e.g., E. coli) and perform one ortwo rounds of selection; and then perform a round of negative selectionagainst a related target (e.g., Candida), taking those phagemids thatfail to bind. These cycles of positive and negative selection are thenrepeated until a population of phages that generally bind to the targetand do not bind to the non-target is generated. This method can beapplied to any pair of microbial strains against which differentialbinding is desired, such as bacteria that are resistant and sensitive toa given antibiotic. This positive/negative enrichment strategy can alsobe used with an antibody-phage display library, which is an even morestandard way to isolate such specific binders.

The directed evolution and selection approach described above also canpotentially be used to generate human antibody fragments or peptidesthat provide the class, subclass and species specificity describedabove.

In some embodiments, at least two microbe surface-binding domains (e.g.,carbohydrate recognition domains), including at least three, at leastfour, at least five, at least six, at least seven, at least eight, atleast nine, at least ten or more microbe surface-binding domains, can belinked together to form a multimeric microbe surface-binding domain orcarbohydrate recognition domain. In such embodiments, the distancesbetween microbe surface-binding domains (e.g., carbohydrate recognitiondomains) can be engineered to match with the distance between thebinding sites on the target microbe surface.

A multimeric microbe surface-binding domain can have each of theindividual microbe surface-binding domains the same. Alternatively, amultimeric microbe surface-binding domain can have at least one, atleast two, or at least three microbe surface-binding domains differentfrom the rest. In such embodiments, microbe surface-binding domains thatshare a common binding specificity for carbohydrates on a microbesurface can be used. By way of example only, the fibrinogen-like domainof several lectins has a similar function to the CRD of C-type lectinsincluding MBL, and function as pattern-recognition receptors todiscriminate pathogens from self. One of such lectins comprising thefibrinogen-like domain is serum ficolins.

Serum ficolins have a common binding specificity for GlcNAc(N-acetylglucosamine), elastin or GalNAc (N-acetyl-galactosamine). Thefibrinogen-like domain is responsible for the carbohydrate binding. Inhuman serum, two types of ficolin, known as L-ficolin (also called P35,ficolin L, ficolin 2 or hucolin) and H-ficolin (also called Hakataantigen, ficolin 3 or thermolabile b2-macroglycoprotein), have beenidentified, and both of them have lectin activity. L-ficolin recognisesGlcNAc and H-ficolin recognises GalNAc. Another ficolin known asM-ficolin (also called P3 5-related protein, ficolin 1 or ficolin A) isnot considered to be a serum protein and is found in leucocytes and inthe lungs. L-ficolin and H-ficolin activate the lectin-complementpathway in association with MASPs. M-Ficolin, L-ficolin and H-ficolinhas calcium-independent lectin activity. Accordingly, in someembodiments, an engineered microbe-targeting, e.g., an engineered MBLmolecule, can comprise MBL and L-ficolin carbohydrate recognitiondomains, MBL and H-ficolin carbohydrate recognition domains, or acombination thereof.

Any art-recognized recombinant carbohydrate-binding proteins orcarbohydrate recognition domains can also be used in the engineeredmicrobe-targeting molecules. For example, recombinant mannose-bindinglectins, e.g., but not limited to, the ones disclosed in the U.S. Pat.Nos. 5,270,199; 6,846,649; and U.S. Patent Application No. US2004/0229212, the contents of which are incorporated herein byreference, can be used in constructing the engineered MBL moleculesdescribed herein.

In one embodiment, the microbe-binding molecule comprises an MBL, acarbohydrate recognition domain of an MBL, or a genetically engineeredversion of MBL (FcMBL) as described in International Application No. WO2011/090954, filed Jan. 19, 2011, the content of both of which isincorporated herein by reference. Amino acid sequences for MBL andengineered MBL include, but are not limited to:

(i) MBL full length (SEQ ID NO. 1): MSLFPSLPLL LLSMVAASYSETVTCEDAQK TCPAVIACSS PGINGFPGKD GRDGTKGEKGEPGQGLRGLQ GPPGKLGPPG NPGPSGSPGP KGQKGDPGKSPDGDSSLAAS ERKALQTEMA RIKKWLTFSL GKQVGNKFFLTNGEIMTFEK VKALCVKFQA SVATPRNAAE NGAIQNLIKEEAFLGITDEK TEGQFVDLTG NRLTYTNWNE GEPNNAGSDEDCVLLLKNGQ WNDVPCSTSH LAVCEFPI (ii)MBL without the signal sequence (SEQ ID NO. 2): ETVTCEDAQKTCPAVIACSS PGINGFPGKD GRDGTKGEKG EPGQGLRGLQGPPGKLGPPG NPGPSGSPGP KGQKGDPGKS PDGDSSLAASERKALQTEMA RIKKWLTFSL GKQVGNKFFL TNGEIMTFEKVKALCVKFQA SVATPRNAAE NGAIQNLIKE EAFLGITDEKTEGQFVDLTG NRLTYTNWNE GEPNNAGSDE DCVLLLKNGQ WNDVPCSTSH LAVCEFPI (iii)Truncated MBL (SEQ ID NO. 3): AASERKALQT EMARIKKWLTFSLGKQVGNK FFLTNGEIMT FEKVKALCVK FQASVATPRNAAENGAIQNL IKEEAFLGIT DEKTEGQFVD LTGNRLTYTNWNEGEPNNAG SDEDCVLLLK NGQWNDVPCS TSHLAVCEFP I (iv)Carbohydrate recognition domain (CRD) of MBL (SEQ ID NO. 4):VGNKFFLTNG EIMTFEKVKA LCVKFQASVA TPRNAAENGAIQNLIKEEAF LGITDEKTEG QFVDLTGNRL TYTNWNEGEPNNAGSDEDCV LLLKNGQWND VPCSTSHLAV CEFPI (v) Neck +Carbohydrate recognition domain of MBL (SEQ ID NO. 5):PDGDSSLAAS ERKALQTEMA RIKKWLTFSL GKQVGNKFFLTNGEIMTFEK VKALCVKFQA SVATPRNAAE NGAIQNLIKEEAFLGITDEK TEGQFVDLTG NRLTYTNWNE GEPNNAGSDEDCVLLLKNGQ WNDVPCSTSH LAVCEFPI (vi) FcMBL.81 (SEQ ID NO. 6):EPKSSDKTHT CPPCPAPELL GGPSVFLFPPKPKDTLMISR TPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSNKALPAPIEKT ISKAKGQPRE PQVYTLPPSR DELTKNQVSLTCLVKGFYPS DIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSPGAPDGDSSLAASERKALQTE MARIKKWLTF SLGKQVGNKFFLTNGEIMTF EKVKALCVKF QASVATPRNA AENGAIQNLIKEEAFLGITD EKTEGQFVDL TGNRLTYTNW NEGEPNNAGSDEDCVLLLKN GQWNDVPCST SHLAVCEFPI (vii) AKT-FcMBL (SEQ ID NO. 7):AKTEPKSSDKTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKFNWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLNGKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSRDELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTPPVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNHYTQKSLSLSP GAPDGDSSLA ASERKALQTE MARIKKWLTFSLGKQVGNKF FLTNGEIMTF EKVKALCVKF QASVATPRNAAENGAIQNLI KEEAFLGITD EKTEGQFVDL TGNRLTYTNWNEGEPNNAGS DEDCVLLLKN GQWNDVPCST SHLAVCEFPI (viii)FcMBL.111 (SEQ ID NO. 8): EPKSSDKTHT CPPCPAPELL GGPSVFLFPPKPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVHNAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSNKALPAPIEKT ISKAKGQPRE PQVYTLPPSR DELTKNQVSLTCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFFLYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSPGATSKQVGNKF FLTNGEIMTF EKVKALCVKF QASVATPRNAAENGAIQNLI KEEAFLGITD EKTEGQFVDL TGNRLTYTNWNEGEPNNAGS DEDCVLLLKN GQWNDVPCST SHLAVCEFPI

In some embodiments, a microbe-binding molecule comprises an amino acidsequence selected from SEQ ID NO. 1-SEQ ID NO. 8.

Without wishing to be bound by a theory, microbe-binding moleculescomprising lectins or modified versions thereof can act asbroad-spectrum pathogen binding molecules. Accordingly, microbes and/ormicrobial matter present in a test sample can be captured usinglectin-based microbe-binding molecules without identifying the microbe.

Linkers

As used herein, the term “linker” generally refers to a molecular entitythat can directly or indirectly connect at two parts of a composition,e.g., at least one microbe surface-binding domain and at least onesubstrate-binding domain. In some embodiments, the linker can directlyor indirectly connect to one or more microbe surface-binding domains.Without limitations, in some embodiments, the linker can also providebinding sites to one or more microbes and/or microbial matter. In suchembodiments, the microbe-binding sites on the linker can bind to thesame types and/or species of microbes as the microbes bind to amicrobe-surface-binding domain. Alternatively or additionally, themicrobe-binding sites on the linker can capture different types and/orspecies of microbes than the ones that bind to a microbe surface-bindingdomain described herein.

Linkers can be configured according to a specific need, e.g., based onat least one of the following characteristics. By way of example only,in some embodiments, linkers can be configured to have a sufficientlength and flexibility such that it can allow for a microbesurface-binding domain to orient accordingly with respect to at leastone carbohydrate on a microbe surface. In some embodiments, linkers canbe configured to allow multimerization of at least two engineeredmicrobe-targeting molecules (e.g., to from a di-, tri-, tetra-, penta-,or higher multimeric complex) while retaining biological activity (e.g.,microbe-binding activity). In some embodiments, linkers can beconfigured to facilitate expression and purification of the engineeredmicrobe-targeting molecule described herein. In some embodiments,linkers can be configured to provide at least one recognition site forproteases or nucleases. In addition, linkers are preferably non-reactivewith the functional components of the engineered molecule describedherein (e.g., minimal hydrophobic or charged character to react with thefunctional protein domains such as a microbe surface-binding domain or asubstrate-binding domain).

In some embodiments, a linker can be configured to have any length in aform of a peptide, peptidomimetic, an aptamer, a protein, a nucleic acid(e.g., DNA or RNA), or any combinations thereof. In some embodiments,the peptidyl or nucleic acid linker can vary from about 1 to about 1000amino acids long, from about 10 to about 500 amino acids long, fromabout 30 to about 300 amino acids long, or from about 50 to about 150amino acids long. Longer or shorter linker sequences can be also usedfor the engineered microbe-targeting molecules described herein. In oneembodiment, the peptidyl linker has an amino acid sequence of about 200to 300 amino acids in length.

In some embodiments, a peptide or nucleic acid linker can be configuredto have a sequence comprising at least one of the amino acids selectedfrom the group consisting of glycine (Gly), serine (Ser), asparagine(Asn), threonine (Thr), methionine (Met) or alanine (Ala), or at leastone of codon sequences encoding the aforementioned amino acids (i.e.,Gly, Ser, Asn, Thr, Met or Ala). Such amino acids and correspondingnucleic acid sequences are generally used to provide flexibility of alinker. However, in some embodiments, other uncharged polar amino acids(e.g., Gln, Cys or Tyr), nonpolar amino acids (e.g., Val, Leu, Ile, Pro,Phe, and Trp), or nucleic acid sequences encoding the amino acidsthereof can also be included in a linker sequence. In alternativeembodiments, polar amino acids or nucleic acid sequence thereof can beadded to modulate the flexibility of a linker. One of skill in the artcan control flexibility of a linker by varying the types and numbers ofresidues in the linker. See, e.g., Perham, 30 Biochem. 8501 (1991);Wriggers et al., 80 Biopolymers 736 (2005).

In alternative embodiments, a linker can be a chemical linker of anylength. In some embodiments, chemical linkers can comprise a direct bondor an atom such as oxygen or sulfur, a unit such as NH, C(O), C(O)NH,SO, SO₂, SO₂NH, or a chain of atoms, such as substituted orunsubstituted C₁-C₆ alkyl, substituted or unsubstituted C₂-C₅ alkenyl,substituted or unsubstituted C₂-C₆ alkynyl, substituted or unsubstitutedC₆-C₁₂ aryl, substituted or unsubstituted C₅-C₁₁ heteroaryl, substitutedor unsubstituted C₅-C₁₂ heterocyclyl, substituted or unsubstitutedC₃-C₁₂ cycloalkyl, where one or more methylenes can be interrupted orterminated by O, S, S(O), SO₂, NH, or C(O). In some embodiments, thechemical linker can be a polymer chain (branched or linear).

In some embodiments where the linker is a peptide, such peptidyl linkercan comprise at least a portion of an immunoglobulin, e.g., IgA, IgD,IgE, IgG and IgM including their subclasses (e.g., IgG1), or a modifiedmolecule or recombinant thereof. In some embodiments, the peptide linkercan comprise a portion of fragment crystallization (Fc) region of animmunoglobulin or a modified thereof. In such embodiments, the portionof the Fc region that can be used as a linker can comprise at least oneregion selected from the group consisting of a hinge region, a CH2region, a CH3 region, and any combinations thereof. By way of example,in some embodiments, a CH2 region can be excluded from the portion ofthe Fc region as a linker. In one embodiment, Fc linker comprises ahinge region, a CH2 domain and a CH3 domain, e.g., Fc IgG 106 as shownin FIG. 1B and FIG. 3. Such Fc linker can be used to facilitateexpression and purification of the engineered microbe-targetingmolecules described herein. The N terminal Fc has been shown to improveexpression levels, protein folding and secretion of the fusion partner.In addition, the Fc has a staphylococcal protein A binding site, whichcan be used for one-step purification protein A affinity chromatography.See Lo K M et al. (1998) Protein Eng. 11: 495-500. Further, the proteinA binding site can be used to facilitate binding of protein A-expressingor protein G-expressing microbes in the absence of calcium ions. Suchbinding capability can be used to develop methods for distinguishingprotein A-expressing microbes (e.g., S. aureus) from non-proteinA-expressing or non-protein G-expressing microbes (e.g., E. coli)present in a test sample, and various embodiments of such methods willbe described in detail later. Further, such Fc linker have a moleculeweight above a renal threshold of about 45 kDa, thus reducing thepossibility of engineered microbe-targeting molecules being removed byglomerular filtration. Additionally, the Fc linker can allowdimerization of two engineered microbe-targeting molecules to form adimer, e.g., the dimeric engineered MBL molecule 100 as shown in FIG.1B.

In some embodiments where the linker comprises a Fc region or a fragmentthereof, the Fc region or a fragment thereof can comprise at least onemutation, e.g., to modify the performance of the engineeredmicrobe-targeting molecules. For example, in some embodiments, ahalf-life of the engineered microbe-targeting molecules described hereincan be increased, e.g., by mutating an amino acid lysine (K) at theresidue 232 of SEQ ID NO. 9 to alanine (A). Other mutations, e.g.,located at the interface between the CH2 and CH3 domains shown in Hintonet al (2004) J Biol Chem. 279:6213-6216 and Vaccaro C. et al. (2005) NatBiotechnol. 23: 1283-1288, can be also used to increase the half-life ofthe IgG1 and thus the engineered microbe-targeting molecules.

In some embodiments, the linker can be albumin, transferrin or afragment thereof. Such linkers can be used to extend the plasmahalf-life of the engineered microbe-targeting molecules and thus aregood for in vivo administration. See Schmidt S R (2009) Curr Opin DrugDiscov Devel. 12: 284.

When the engineered microbe-targeting molecules are used as therapeuticsin vivo, the linker can be further modified to modulate the effectorfunction such as antibody-dependent cellular cytotoxicity (ADCC) andcomplement-dependent cytotoxicity (CDC). By way of example only, the Fcregion for use as a linker can mediate ADCC and CDC. In ADCC, the Fcregion can generally bind to Fc receptors on the surface of immuneeffector cells such as natural killers and macrophages, leading to thephagocytosis or lysis of a targeted cell. In CDC, the Fc region cangenerally trigger the complement cascade at the cell surface to kill thetargeted cell. Accordingly, modulating effector functions can beachieved by engineering the Fc region to either increase or decreasetheir binding to the Fc receptors on the surface of the immune effectorcells or the complement factors. For example, numerous mutations withina Fc region for modulating ADCC and CDC are well known to a skilledartisan, e.g., see Armour K L. et al. (1999) Eur J Immmunol 29:2613-2624; Shields R L. et al. (2001) J Biol Chem. 276: 6591-6604;Idusogie E E. et al. (2001) J Immunol. 166: 2571-2575; Idusogie E E. etal. (2000) J Immunol. 155: 1165-1174; and Steurer W. et al. (1995) JImmunol. 155: 1165-1674. In one embodiment, the amino acid asparagine(N) at the residue 82 of the SEQ ID NO. 6 can be mutated to asparticacid (D), e.g., to remove the glycosylation of Fc and thus, in turn,reduce ADCC and CDC functions.

In various embodiments, the N-terminus or the C-terminus of the linker,e.g., the portion of the Fc region, can be modified. By way of exampleonly, the N-terminus or the C-terminus of the linker can be extended byat least one additional linker described herein, e.g., to providefurther flexibility, or to attach additional molecules. In someembodiments, the N-terminus of the linker can be linked directly orindirectly (via an additional linker) with a substrate-binding domainadapted for orienting the carbohydrate recognition domain away from thesubstrate.

In some embodiments, the linker can be embodied as part of the microbesurface-binding domain, or part of the carbohydrate-binding protein(e.g., MBL and/or the neck region thereof).

In some embodiments, the linker can be a physical substrate, e.g.,microparticles or magnetic microbes, to which a plurality of microbesurface-binding domains (including carbohydrate recognition domain) canbind, provided that there is at least a certain distance between themicrobe surface-binding domain and the substrate surface sufficient forthe microbe surface-binding domain to interact effectively withmicrobes. In some embodiments, the distance between the microbesurface-binding domain and the substrate can range from about 50angstroms to about 5000 angstroms, from about 100 angstroms to about2500 angstroms, or from about 200 angstroms to about 1000 angstroms.

The linkers can be of any shape. In some embodiments, the linkers can belinear. In some embodiments, the linkers can be folded. In someembodiments, the linkers can be branched. For branched linkers, eachbranch of a microbe surface-binding domain can comprise at least onemicrobe surface-binding domain. In other embodiments, the linker adoptsthe shape of the physical substrate.

In some embodiments provided herein, the linker can further comprise adetectable label. In some embodiments, the detectable label can be achromogenic or fluorogenic microbe enzyme substrate so that when amicrobe binds to the engineered microbe-targeting molecule, the enzymethat the microbe releases can interact with the detectable label toinduce a color change. Examples of such microbe enzyme substrate caninclude, but are not limited to, indoxyl butyrate, indoxyl glucoside,esculin, magneta glucoside, red-β-glucuronide,2-methoxy-4-(2-nitrovinyl) phenyl β-D-glu-copyranoside,2-methoxy-4-(2-nitrovinyl) phenyl β-D-cetamindo-2-deoxyglucopyranoside,and any other art-recognized microbe enzyme substrates. Such embodimentscan act as an indicator for the presence of a microbe or pathogen.

Conjugation of Engineered Microbe-Targeting Molecules to a Substrate

The engineered microbe-targeting molecules can be immobilized on anysubstrate for various applications and/or purposes. For example, whenthe affinity of a single microbe surface-binding domain for a targetmolecule (e.g., a carbohydrate recognition domain for asugar/carbohydrate molecule) is relatively low, and such binding isgenerally driven by avidity and multivalency, multivalency of suchengineered microbe-targeting molecules can be effectively increased byattachment of a plurality of the engineered microbe-targeting molecules(e.g., each with one or two or more carbohydrate recognition domains) toa solid substrate (e.g., a nanometer- or micrometer-sized bead) at ahigh density, which can be varied to provide optimal functionality.Alternatively, the engineered microbe-targeting molecules can beimmobilized on a solid substrate for easy handling during usage, e.g.,for isolation, observation or microscopic imaging.

The attachment of the engineered microbe-binding molecule (e.g., FcMBL)to a substrate surface (e.g., membrane surface, glass surface, tubingsurface) can be performed with multiple approaches, for example, bydirect cross-linking the engineered microbe-binding molecule (e.g.,FcMBL) to the substrate surface; cross-linking the engineeredmicrobe-binding molecule (e.g., FcMBL) to the substrate surface via anucleic acid matrix (e.g., DNA matrix or DNA/oligonucleotide origamistructures) for orientation and concentration to increase detectionsensitivity; cross-linking FcMBL to the substrate surface via adendrimer-like structure (e.g., PEG/Chitin-structure) to increasedetection sensitivity; attracting FcMBL-coated magnetic microbeads tothe substrate surface with a focused magnetic field gradient applied tothe substrate surface, attaching an engineered microbe-binding molecule(e.g., FcMBL) to a substrate via biotin-avidin or biotin-avidin-likeinteraction, or any other art-recognized methods.

For engineered microbe-targeting molecules or mannose-binding lectinmolecules to be immobilized on or conjugated to a substrate, theengineered molecules described herein can further comprise at least one(e.g., one, two, three, four, five, six, seven, eight, nine, ten,eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen,eighteen, nineteen, twenty or more) substrate-binding domain, e.g.,adapted for orienting the carbohydrate recognition domain away from thesubstrate. Without limitations, exemplary types of substrates can be anucleic acid scaffold, a biological molecule (e.g., a living cell), or asolid surface. In some embodiments, the solid surface can befunctionalized with a coupling molecule, e.g., an amino group, tofacilitate the conjugation of engineered microbe surface-binding domainsto the solid surface.

As used herein, the term “substrate-binding domain” refers to anymolecule that facilitates the conjugation of the engineered moleculesdescribed herein to a substrate or a functionalized substrate. In someembodiments, the substrate-binding domain can comprise at least oneamino group that can non-covalently or covalently coupled withfunctional groups on the surface of the substrate. For example, theprimary amines of the amino acid residues (e.g., lysine or cysteineresidues) at the N-terminus or in close proximity to the N-terminus ofthe engineered microbe surface-binding domains (e.g., engineeredmannose-binding lectins) can be used to couple with functional groups onthe substrate surface.

In some embodiments, the substrate-binding domain can comprise at leastone, at least two, at least three or more oligopeptides. The length ofthe oligonucleotide can vary from about 2 amino acid residues to about10 amino acid residues, or about 2 amino acid residues to about 5 aminoacid residues. Determination of an appropriate amino acid sequence ofthe oligonucleotide for binding with different substrates is well withinone of skill in the art. For example, as shown in FIG. 1B, according toone or more embodiments, the substrate-binding domain 108 can comprisean oligopeptide comprising an amino acid sequence of AKT, which providesa single biotinylation site for subsequent binding tostreptavidin-coated substrate, e.g., a magnetic microbead 110. Suchsingle biotinylation site can also enable the carbohydrate recognitiondomain of an engineered microbe surface-binding domain to orient awayfrom the substrate, and thus become more accessible to microbes orpathogens. See, e.g., Witus et al. (2010) JACS 132: 16812.

In some embodiments, the substrate-binding domain can comprise at leastone oligonucleotide. The sequence and length of the oligonucleotides canbe configured according to the types of the substrate, binding density,and/or desired binding strength. For example, if the substrate is anucleic acid scaffold, e.g., a DNA scaffold, the oligonucleotidesequence of the substrate-binding domain can be designed such that it iscomplementary to a sub-sequence of the nucleic acid scaffold to wherethe substrate-binding domain can hybridize.

In some embodiments, the oligonucleotides can include aptamers. As usedherein, the term “aptamer” means a single-stranded, partiallysingle-stranded, partially double-stranded or double-stranded nucleotidesequence capable of specifically recognizing a selectednon-oligonucleotide molecule or group of molecules by a mechanism otherthan Watson-Crick base pairing or triplex formation. Aptamers caninclude, without limitation, defined sequence segments and sequencescomprising nucleotides, ribonucleotides, deoxyribonucleotides,nucleotide analogs, modified nucleotides and nucleotides comprisingbackbone modifications, branchpoints and nonnucleotide residues, groupsor bridges. Methods for selecting aptamers for binding to a molecule arewidely known in the art and easily accessible to one of ordinary skillin the art. The oligonucleotides including aptamers can be of anylength, e.g., from about 1 nucleotide to about 100 nucleotides, fromabout 5 nucleotides to about 50 nucleotides, or from about 10nucleotides to about 25 nucleotides. Generally, a longer oligonucleotidefor hybridization to a nucleic acid scaffold can generate a strongerbinding strength between the engineered microbe surface-binding domainand substrate.

Alternatively or additionally, the surface of a substrate can befunctionalized to include coupling molecules described herein. As usedherein, the term “coupling molecule” refers to any molecule or anyfunctional group that is capable of selectively binding with anengineered microbe surface-binding domain described herein.Representative examples of coupling molecules include, but are notlimited to, antibodies, antigens, lectins, proteins, peptides, nucleicacids (DNA, RNA, PNA and nucleic acids that are mixtures thereof or thatinclude nucleotide derivatives or analogs); receptor molecules, such asthe insulin receptor; ligands for receptors (e.g., insulin for theinsulin receptor); and biological, chemical or other molecules that haveaffinity for another molecule, such as biotin and avidin. The couplingmolecules need not comprise an entire naturally occurring molecule butmay consist of only a portion, fragment or subunit of a naturally ornon-naturally occurring molecule, as for example the Fab fragment of anantibody. The coupling molecule can further comprise a detectable label.The coupling molecule can also encompass various functional groups thatcan couple the substrate to the engineered microbe surface-bindingdomains. Examples of such functional groups include, but are not limitedto, an amino group, a carboxylic acid group, an epoxy group, and a tosylgroup.

In some embodiments, the engineered microbe-targeting molecule can beconjugated to a substrate surface through a covalent or non-covalentinteraction. The engineered microbe-targeting molecule and/or couplingmolecule can be conjugated to the surface of a solid substratecovalently or non-covalently using any of the methods known to those ofskill in the art. For example, covalent immobilization can beaccomplished through, for example, silane coupling. See, e.g., Weetall,15 Adv. Mol. Cell Bio. 161 (2008); Weetall, 44 Meths. Enzymol. 134(1976). The covalent interaction between the engineeredmicrobe-targeting molecule and/or coupling molecule and the surface canalso be mediated by other art-recognized chemical reactions, such as NHSreaction or a conjugation agent. The non-covalent interaction betweenthe engineered microbe-targeting molecule and/or coupling molecule andthe surface can be formed based on ionic interactions, van der Waalsinteractions, dipole-dipole interactions, hydrogen bonds, electrostaticinteractions, and/or shape recognition interactions.

Without limitations, conjugation can include either a stable or a labile(e.g. cleavable) bond or conjugation agent. Exemplary conjugationsinclude, but are not limited to, covalent bond, amide bond, additions tocarbon-carbon multiple bonds, azide alkyne Huisgen cycloaddition,Diels-Alder reaction, disulfide linkage, ester bond, Michael additions,silane bond, urethane, nucleophilic ring opening reactions: epoxides,non-aldol carbonyl chemistry, cycloaddition reactions: 1,3-dipolarcycloaddition, temperature sensitive, radiation (IR, near-IR, UV)sensitive bond or conjugation agent, pH-sensitive bond or conjugationagent, non-covalent bonds (e.g., ionic charge complex formation,hydrogen bonding, pi-pi interactions, cyclodextrin/adamantly host guestinteraction) and the like.

As used herein, the term “conjugation agent” means an organic moietythat connects two parts of a compound. Linkers typically comprise adirect bond or an atom such as oxygen or sulfur, a unit such as NR′,C(O), C(O)NH, SO, SO₂, SO₂NH or a chain of atoms, such as substituted orunsubstituted alkyl, substituted or unsubstituted alkenyl, substitutedor unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl,heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl,heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl,heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl,alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl,alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl,alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl,alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl,alkenylheteroarylalkenyl, alkenylheteroarylalkynyl,alkynylheteroarylalkyl, alkynylheteroarylalkenyl,alkynylheteroarylalkynyl, alkylheterocyclylalkyl,alkylheterocyclylalkenyl, alkylhererocyclylalkynyl,alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl,alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl,alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl,alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl,alkynylhereroaryl, where one or more methylenes can be interrupted orterminated by O, S, S(O), SO₂, NH, C(O)N(R¹)₂, C(O), cleavable linkinggroup, substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted heterocyclic; where R¹ ishydrogen, acyl, aliphatic or substituted aliphatic.

Without limitations, any conjugation chemistry known in the art forconjugating two molecules or different parts of a composition togethercan be used for linking at least one engineered microbe-targetingmolecule to a substrate. Exemplary coupling molecules and/or functionalgroups for conjugating at least one engineered microbe-targetingmolecule to a substrate include, but are not limited to, a polyethyleneglycol (PEG, NH₂-PEG_(X)-COOH which can have a PEG spacer arm of variouslengths X, where 1<X<100, e.g., PEG-2K, PEG-5K, PEG-10K, PEG-12K,PEG-15K, PEG-20K, PEG-40K, and the like), maleimide conjugation agent,PASylation, HESylation, Bis(sulfosuccinimidyl) suberate conjugationagent, DNA conjugation agent, peptide conjugation agent, silaneconjugation agent, polysaccharide conjugation agent, hydrolyzableconjugation agent, and any combinations thereof.

In alternative embodiments, the engineered microbe surface-bindingdomains or the engineered microbe-targeting molecule can be conjugatedonto the surface of the solid substrate by a coupling molecule pair. Theterms “coupling molecule pair” and “coupling pair” as usedinterchangeably herein refer to the first and second molecules thatspecifically bind to each other. One member of the binding pair isconjugated with the solid substrate while the second member isconjugated with the substrate-binding domain of an engineered microbesurface-binding domain. As used herein, the phrase “first and secondmolecules that specifically bind to each other” refers to binding of thefirst member of the coupling pair to the second member of the couplingpair with greater affinity and specificity than to other molecules.

Exemplary coupling molecule pairs include, without limitations, anyhaptenic or antigenic compound in combination with a correspondingantibody or binding portion or fragment thereof (e.g., digoxigenin andanti-digoxigenin; mouse immunoglobulin and goat antimouseimmunoglobulin) and nonimmunological binding pairs (e.g., biotin-avidin,biotin-streptavidin), hormone (e.g., thyroxine and cortisol-hormonebinding protein), receptor-receptor agonist, receptor-receptorantagonist (e.g., acetylcholine receptor-acetylcholine or an analogthereof), IgG-protein A, lectin-carbohydrate, enzyme-enzyme cofactor,enzyme-enzyme inhibitor, and complementary oligonucleotide pairs capableof forming nucleic acid duplexes). The coupling molecule pair can alsoinclude a first molecule that is negatively charged and a secondmolecule that is positively charged.

One example of using coupling pair conjugation is the biotin-avidin orbiotin-streptavidin conjugation. In this approach, one of the members ofthe coupling pair (e.g., a portion of the engineered microbe-targetingmolecule such as substrate-binding domain, or a substrate) isbiotinylated and the other (e.g., a substrate or the engineeredmicrobe-targeting molecule) is conjugated with avidin or streptavidin.Many commercial kits are also available for biotinylating molecules,such as proteins. For example, an aminooxy-biotin (AOB) can be used tocovalently attach biotin to a molecule with an aldehyde or ketone group.In one embodiment, AOB is attached to the substrate-binding domain(e.g., comprising AKT oligopeptide) of the engineered microbe-targetingmolecule.

One non-limiting example of using conjugation with a coupling moleculepair is the biotin-sandwich method. See, e.g., Davis et al., 103 PNAS8155 (2006). The two molecules to be conjugated together arebiotinylated and then conjugated together using tetravalentstreptavidin. In addition, a peptide can be coupled to the 15-amino acidsequence of an acceptor peptide for biotinylation (referred to as AP;Chen et al., 2 Nat. Methods 99 (2005)). The acceptor peptide sequenceallows site-specific biotinylation by the E. coli enzyme biotin ligase(BirA; Id.). An engineered microbe surface-binding domain can besimilarly biotinylated for conjugation with a solid substrate. Manycommercial kits are also available for biotinylating proteins. Anotherexample for conjugation to a solid surface would be to use PLP-mediatedbioconjugation. See, e.g., Witus et al., 132 JACS 16812 (2010). Asdescribed earlier, an AKT sequence on the N terminal of the engineeredmicrobe-targeting molecule (e.g., N terminal of the linker between thesubstrate binding domain and the carbohydrate-binding molecule such asFc region as described earlier) can allow the substrate binding domainto be biotinylated at a single site and further conjugated to thestreptavidin-coated solid surface.

Still another example of using coupling pair conjugation isdouble-stranded nucleic acid conjugation. In this approach, one of themembers of the coupling pair (e.g., a portion of the engineeredmicrobe-targeting molecule such as substrate-binding domain, or asubstrate) can be conjugated with a first strand of the double-strandednucleic acid and the other (e.g., a substrate, or an engineeredmicrobe-targeting molecule) is conjugated with the second strand of thedouble-stranded nucleic acid. Nucleic acids can include, withoutlimitation, defined sequence segments and sequences comprisingnucleotides, ribonucleotides, deoxyribonucleotides, nucleotide analogs,modified nucleotides and nucleotides comprising backbone modifications,branchpoints and nonnucleotide residues, groups or bridges.

In some embodiments, the linker can comprise at least one cleavablelinking group. A cleavable linking group is one which is sufficientlystable under one set of conditions, but which is cleaved under adifferent set of conditions to release the two parts the linker isholding together. In some embodiments, the cleavable linking group iscleaved at least 10 times or more, e.g., at least 100 times faster undera first reference condition (which can, e.g., be selected to mimic orrepresent a microbe-infected condition, such as a microbe-infectedtissue or body fluid, or a microbial biofilm occurring in anenvironment) than under a second reference condition (which can, e.g.,be selected to mimic or represent non-infected conditions, e.g., foundin the non-infected blood or serum, or in an non-infected environment).

Cleavable linking groups are susceptible to cleavage agents, e.g.,hydrolysis, pH, redox potential or the presence of degradativemolecules. Generally, cleavage agents are more prevalent or found athigher levels or activities at a site of interest (e.g. a microbialinfection) than in non-infected area. Examples of such degradativeagents include: redox agents which are selected for particularsubstrates or which have no substrate specificity, including, e.g.,oxidative or reductive enzymes or reductive agents such as mercaptans,present in cells, that can degrade a redox cleavable linking group byreduction; esterases; amidases; endosomes or agents that can create anacidic environment, e.g., those that result in a pH of five or lower;enzymes that can hydrolyze or degrade an acid cleavable linking group byacting as a general acid, peptidases (which can be substrate specific)and proteases, and phosphatases.

A linker can include a cleavable linking group that is cleavable by aparticular enzyme. The type of cleavable linking group incorporated intoa linker can depend on the cell, organ, or tissue to be targeted. Insome embodiments, cleavable linking group is cleaved at least 1.25, 1.5,1.75, 2, 3, 4, 5, 10, 25, 50, or 100 times faster under a firstreference condition (or under in vitro conditions selected to mimic amicrobe-infected condition, such as a microbe-infected tissue or bodyfluid, or a microbial biofilm occurring in an environment or on aworking surface) than under a second reference condition (or under invitro conditions selected to mimic non-infected conditions, e.g., foundin the non-infected blood or serum, or in an non-infected environment).In some embodiments, the cleavable linking group is cleaved by less than90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or 1% in thenon-infected conditions, e.g., found in the non-infected blood or serum,or in an non-infected environment, as compared to a microbe-infectedcondition, such as a microbe-infected tissue or body fluid, or amicrobial biofilm occurring in an environment or on a working surface.

Exemplary cleavable linking groups include, but are not limited to,hydrolyzable linkers, redox cleavable linking groups (e.g., —S—S— and—C(R)₂—S—S—, wherein R is H or C₁-C₆ alkyl and at least one R is C₁-C₆alkyl such as CH₃ or CH₂CH₃); phosphate-based cleavable linking groups(e.g., —O—P(O)(OR)—O—, —O—P(S)(OR)—O—, —O—P(S)(SR)—O—, —S—P(O)(OR)—O—,—O—P(O)(OR)—S—, —S—P(O)(OR)—S—, —O—P(S)(ORk)-S—, —S—P(S)(OR)—O—,—O—P(O)(R)—O—, —O—P(S)(R)—O—, —S—P(O)(R)—O—, —S—P(S)(R)—O—,—S—P(O)(R)—S—, —O—P(S)(R)—S—, —O—P(O)(OH)—O—, —O—P(S)(OH)—O—,—O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O—P(O)(OH)—S—, —S—P(O)(OH)—S—,—O—P(S)(OH)—S—, —S—P(S)(OH)—O—, —O—P(O)(H)—O—, —O—P(S)(H)—O—,—S—P(O)(H)—O—, —S—P(S)(H)—O—, —S—P(O)(H)—S—, and —O—P(S)(H)—S—, whereinR is optionally substituted linear or branched C₁-C₁₀ alkyl); acidcleavable linking groups (e.g., hydrazones, esters, and esters of aminoacids, —C═NN— and —OC(O)—); ester-based cleavable linking groups (e.g.,—C(O)O—); peptide-based cleavable linking groups, (e.g., linking groupsthat are cleaved by enzymes such as peptidases and proteases in cells,e.g., —NHCHR^(A)C(O)NHCHR^(B)C(O)—, where R^(A) and R^(B) are the Rgroups of the two adjacent amino acids). A peptide based cleavablelinking group comprises two or more amino acids. In some embodiments,the peptide-based cleavage linkage comprises the amino acid sequencethat is the substrate for a peptidase or a protease. In someembodiments, an acid cleavable linking group is cleavable in an acidicenvironment with a pH of about 6.5 or lower (e.g., about 6.5, 6.0, 5.5,5.0, or lower), or by agents such as enzymes that can act as a generalacid.

Activation agents can be used to activate the components to beconjugated together (e.g., surface of a substrate). Without limitations,any process and/or reagent known in the art for conjugation activationcan be used. Exemplary surface activation method or reagents include,but are not limited to, 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimidehydrochloride (EDC or EDAC), hydroxybenzotriazole (HOBT),N-Hydroxysuccinimide (NHS),2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate methanaminium (HATU), silanization, surfaceactivation through plasma treatment, and the like.

Again, without limitations, any art known reactive group can be used forcoupling. For example, various surface reactive groups can be used forsurface coupling including, but not limited to, alkyl halide, aldehyde,amino, bromo or iodoacetyl, carboxyl, hydroxyl, epoxy, ester, silane,thiol, and the like.

Exemplary Microbe-Targeting Substrates or Products and ApplicationsThereof

Some embodiments of the engineered microbe-targeting molecules describedherein can be immobilized or conjugated to a surface of varioussubstrates. Accordingly, a further aspect provided herein is a“microbe-targeting substrate” or product for targeting or bindingmicrobes comprising a substrate and at least one engineeredmicrobe-targeting molecule described herein, wherein the substratecomprises on its surface at least one, including at least two, at leastthree, at least four, at least five, at least ten, at least 25, at least50, at least 100, at least 250, at least 500, or more engineeredmicrobe-targeting molecules. In some embodiments, the substrate can beconjugated or coated with at least one engineered microbe-targetingmolecule, e.g., an engineered mannose-binding lectin as describedherein, using any of conjugation methods described earlier or any otherart-recognized methods. The terms “microbe-targeting substrate” and“microbe-binding substrate” are used interchangeably herein.

The solid substrate can be made from a wide variety of materials and ina variety of formats. For example, the solid substrate can be utilizedin the form of beads (including polymer microbeads, magnetic microbeads,and the like), filters, fibers, screens, mesh, tubes, hollow fibers,scaffolds, plates, channels, other substrates commonly utilized in assayformats, and any combinations thereof. Examples of substrates include,but are not limited to, nucleic acid scaffolds, protein scaffolds, lipidscaffolds, dendrimers, microparticles or microbeads, nanotubes,microtiter plates, medical apparatuses (e.g., needles or catheters) orimplants, dipsticks or test strips, microchips, filtration devices ormembranes, diagnostic strips, hollow-fiber reactors, microfluidicdevices, living cells and biological tissues or organs, extracorporealdevices, mixing elements (e.g., spiral mixers).

The solid substrate can be made of any material, including, but notlimited to, metal, metal alloy, polymer, plastic, paper, glass, fabric,packaging material, biological material such as cells, tissues,hydrogels, proteins, peptides, nucleic acids, and any combinationsthereof.

The particular format and/or material of the solid substrate depend onthe assay application such as separation/detection methods employed inthe assay. In some embodiments, the format and/or material of the solidsubstrate can be chosen or modified to maximize signal-to-noise ratios,e.g., to minimize background binding, and/or for ease of separation ofreagents and cost. For example, the surface of the solid substrate canbe treated or modified with surface chemistry to minimize chemicalagglutination and non-specific binding. In some embodiments, at least aportion of the substrate surface that is in contact with a test samplecan be treated to become less adhesive to any molecules (includingmicrobes, if any) present in the test sample. By way of example only,the substrate surface in contact with a test sample can be silanized orcoated with a polymer such that the substrate surface is inert to themolecules present in the test sample, including but not limited to,cells or fragments thereof (including blood cells and blood components),proteins, nucleic acids, peptides, small molecules, therapeutic agents,microbes, microorganisms and any combinations thereof. In otherembodiments, a substrate surface can be treated with an omniphobiclayer, which can allow binding of a microbe by the engineeredmicrobe-targeting molecule without a subsequent hydrophobic bindingbetween the microbe and the substrate surface. See, e.g., Wong T S etal., “Bioinspired self-repairing slippery surfaces with pressure-stableomniphobicity.” (2011) Nature 477 (7365): 443-447, and InternationalApplication No.: PCT/US12/21928, the content of which is incorporatedherein by reference, for methods to produce a slippery substratesurface. Accordingly, non-specific binding of molecules from the testsample (including microbes and/or microbial matter) to a substratesurface can be reduced, thus increasing the sensitivity of the microbialdetection.

In some embodiments, the solid substrate can be fabricated from orcoated with a biocompatible material. As used herein, the term“biocompatible material” refers to any material that does notdeteriorate appreciably and does not induce a significant immuneresponse or deleterious tissue reaction, e.g., toxic reaction orsignificant irritation, over time when implanted into or placed adjacentto the biological tissue of a subject, or induce blood clotting orcoagulation when it comes in contact with blood. Suitable biocompatiblematerials include, for example, derivatives and copolymers ofpolyimides, poly(ethylene glycol), polyvinyl alcohol, polyethyleneimine,and polyvinylamine, polyacrylates, polyamides, polyesters,polycarbonates, and polystyrenes. In some embodiments, biocompatiblematerials can include metals, such as titanium and stainless steel, orany biocompatible metal used in medical implants. In some embodiments,biocompatible materials can include paper substrate, e.g., as asubstrate for a diagnostic strip. In some embodiments, biocompatiblematerials can include peptides or nucleic acid molecules, e.g., anucleic acid scaffold such as a 2-D DNA sheet or 3-D DNA scaffold.

Additional material that can be used to fabricate or coat a solidsubstrate include, without limitations, polydimethylsiloxane, polyimide,polyethylene terephthalate, polymethylmethacrylate, polyurethane,polyvinylchloride, polystyrene polysulfone, polycarbonate,polymethylpentene, polypropylene, polyvinylidine fluoride, polysilicon,polytetrafluoroethylene, polysulfone, acrylonitrile butadiene styrene,polyacrylonitrile, polybutadiene, poly(butylene terephthalate),poly(ether sulfone), poly(ether ether ketones), poly(ethylene glycol),styrene-acrylonitrile resin, poly(trimethylene terephthalate), polyvinylbutyral, polyvinylidenedifluoride, poly(vinyl pyrrolidone), and anycombination thereof.

In various embodiments, the substrate can be functionalized with variouscoupling molecules as described earlier.

As used herein, by the “coating” or “coated” is generally meant a layerof molecules or material formed on an outermost or exposed layer of asubstrate surface. With respect to a coating of engineeredmicrobe-targeting molecules on a substrate, the term “coating” or“coated” refers to a layer of engineered microbe-targeting moleculesformed on an outermost or exposed layer of a substrate surface. In someembodiments, the substrate surface can encompass an outer substratesurface and/or an inner substrate surface, e.g., with respect to ahollow structure. For example, the inner surface of a needle or cathetercan be coated with the engineered microbe-targeting molecules describedherein, e.g., for removing any potential microbe contaminants from afluid before administering the fluid to a subject.

The amount of the engineered microbe-targeting molecules conjugated toor coating on a substrate surface can vary with a number of factors suchas a substrate surface area, conjugation/coating density, types ofengineered microbe-targeting molecules, and/or binding performance. Askilled artisan can determine the optimum density of engineeredmicrobe-targeting molecules on a substrate surface using any methodsknown in the art. By way of example only, for magnetic microbeads(including nanobeads) as a substrate (as discussed in detail later), theamount of the engineered microbe-targeting molecules used forconjugating to or coating magnetic microbeads can vary from about 1 wt %to about 30 wt %, or from about 5 wt % to about 20 wt %. In someembodiments, the amount of the engineered microbe-targeting moleculesused for conjugating to or coating magnetic microbeads can be higher orlower, depending on a specific need. However, it should be noted that ifthe amount of the engineered microbe-targeting molecules used forconjugating to or coating the magnetic microbeads is too low, themagnetic microbeads can show a lower binding performance with apathogen/microbe. On the contrary, if the amount of the engineeredmicrobe-targeting molecules used for conjugating to or coating themagnetic microbeads is too high, the dense layer of the engineeredmicrobe-targeting molecules can exert an adverse influence on themagnetic properties of the magnetic microbeads, which in turn candegrade the efficiency of separating the magnetic microbeads from afluid utilizing the magnetic field gradient.

Microbe-Targeting Microparticles:

Some embodiments described herein provide a microbe-targetingmicroparticle comprising at least one engineered microbe-targetingmolecule on its surface. The term “microparticle” as used herein refersto a particle having a particle size of about 0.001 μm to about 100 μm,about 0.005 μm to about 50 μm, about 0.01 μm to about 25 μm, about 0.05μm to about 10 μm, or about 0.05 μm to about 5 μm. In one embodiment,the microparticle has a particle size of about 0.05 μm to about 1 μm. Inone embodiment, the microparticle is about 0.09 μm-about 0.2 μm in size.It will be understood by one of ordinary skill in the art thatmicroparticles usually exhibit a distribution of particle sizes aroundthe indicated “size.” Unless otherwise stated, the term “size” as usedherein refers to the mode of a size distribution of microparticles,i.e., the value that occurs most frequently in the size distribution.Methods for measuring the microparticle size are known to a skilledartisan, e.g., by dynamic light scattering (such as photocorrelationspectroscopy, laser diffraction, low-angle laser light scattering(LALLS), and medium-angle laser light scattering (MALLS)), lightobscuration methods (such as Coulter analysis method), or othertechniques (such as rheology, and light or electron microscopy).

The microparticles can be of any shape, e.g., a sphere. In someembodiments, the term “microparticle” as used herein can encompass amicrosphere. The term “microsphere” as used herein refers to amicroparticle having a substantially spherical form. A substantiallyspherical microparticle is a microparticle with a difference between thesmallest radii and the largest radii generally not greater than about40% of the smaller radii, and more typically less than about 30%, orless than 20%. In one embodiment, the term “microparticle” as usedherein encompasses a microcapsule. The term “microcapsule” as usedherein refers to a microscopic capsule that contains an activeingredient, e.g., a therapeutic agent.

Accordingly, in some embodiments, the microparticles comprising on theirsurface engineered microbe-targeting molecules can encapsulate at leastone active ingredient therein, e.g., a therapeutic agent to treat aninfection, and be used as a cell-targeted drug delivery device. In suchembodiments, the microparticles can comprise biocompatible polymers asdescribed herein. In some embodiments, the microparticles can furthercomprise biodegradable polymers, e.g., for releasing the encapsulateddrugs.

As used herein, the term “biodegradable” refers to the ability of acomposition to erode or degrade in vivo to form smaller chemicalfragments. Degradation can occur, for example, by enzymatic, chemical orphysical processes. Non-limiting examples of biodegradable polymers thatcan be used in aspects provided herein include poly(lactide)s,poly(glycolide)s, poly(lactic acid)s, poly(glycolic acid)s,poly(lactide-co-glycolide), polyanhydrides, polyorthoesters,polycaprolactone, polyesteramides, polycarbonate, polycyanoacrylate,polyurethanes, polyacrylate, blends and copolymers thereof.

Other additional biodegradable polymers include biodegradablepolyetherester copolymers. Generally speaking, the polyetherestercopolymers are amphiphilic block copolymers that include hydrophilic(for example, a polyalkylene glycol, such as polyethylene glycol) andhydrophobic blocks (for example, polyethylene terephthalate). Anexemplary block copolymer is, but is not limited to, poly(ethyleneglycol)-based and poly(butylene terephthalate)-based blocks (PEG/PBTpolymer). PEG/PBT polymers are commercially available from OctoPlus Inc,under the trade designation PolyActive™. Non-limiting examples ofbiodegradable copolymers or multiblock copolymers include the onesdescribed in U.S. Pat. Nos. 5,980,948 and 5,252,701, the contents ofwhich are incorporated herein by reference.

Other biodegradable polymer materials include biodegradableterephthalate copolymers that include a phosphorus-containing linkage.Polymers having phosphoester linkages, called poly(phosphates),poly(phosphonates) and poly(phosphites), are known in the art. See, forexample, Penczek et al., Handbook of Polymer Synthesis, Chapter 17:“Phosphorus-Containing Polymers,” 1077-1 132 (Hans R. Kricheldorf ed.,1992), as well as U.S. Pat. Nos. 6,153,212; 6,485,737; 6,322,797;6,600,010; 6,419,709; 6,419,709; 6,485,737; 6,153,212; 6,322,797 and6,600,010, the contents of which are incorporated herein by reference.

Biodegradable polyhydric alcohol esters can also be used as a materialof a substrate (e.g., a microparticle) (See U.S. Pat. No. 6,592,895,which is incorporated herein by reference). In some embodiments, thebiodegradable polymer can be a three-dimensional crosslinked polymernetwork containing hydrophobic and hydrophilic components which forms ahydrogel with a crosslinked polymer structure, such as the one describedin U.S. Pat. No. 6,583,219. In yet further embodiments, thebiodegradable polymer can comprise a polymer based upon α-amino acids(such as elastomeric copolyester amides or copolyester urethanes, asdescribed in U.S. Pat. No. 6,503,538, which is incorporated herein byreference).

In general, any biocompatible material well known in the art forfabrication of microparticles can be used in embodiments of themicroparticle described herein. Accordingly, a microparticle comprisinga lipidic microparticle core is also within the scope described herein.An exemplary lipidic microparticle core is, but is not limited to, aliposome. A liposome is generally defined as a particle comprising oneor more lipid bilayers enclosing an interior, e.g., an aqueous interior.In one embodiment, a liposome can be a vesicle formed by a bilayer lipidmembrane. Methods for the preparation of liposomes are well described inthe art, e.g., Szoka and Papahadjopoulos (1980) Ann. Rev. Biophys.Bioeng. 9: 467, Deamer and Uster (1983) Pp. 27-51 In: Liposomes, ed. M.J. Ostro, Marcel Dekker, New York.

Microbe-Targeting Magnetic Microbeads:

In some particular embodiments, provided herein is a “microbe-targetingmagnetic microbead” wherein a magnetic microbead comprising on itssurface at least one engineered microbe-targeting molecule, e.g., anengineered mannose-binding lectin as described herein. By way of exampleonly, a microbe targeting magnetic microbead 112, as shown in FIG. 1C,can comprise a magnetic microbead 110 coated with a plurality of themicrobe-targeting molecules, e.g., dimeric microbe-targeting molecules100. Such microbe-targeting magnetic microbeads can be used to separatemicrobes or pathogens from a test sample, e.g., but not limited to, anyfluid, including a biological fluid such as blood. In some embodiments,the microbe-targeting magnetic microbeads can be used to remove livingmicrobes or pathogens. Using magnetic microbeads as a substrate can beadvantageous because the microbe-bound magnetic microbeads can be easilyseparated from a sample fluid using a magnetic field gradient, beexamined for the presence of the microbe, and/or be used to transfer thecollected microbes to conventional pathogen culture and sensitivitytesting assays. Thus, in some embodiments, the microbe-targetingmagnetic microbeads can be used to remove microbe contaminants from anysource or in any fluid, e.g., a biological fluid (e.g., blood sample),environmental fluid or surface (e.g., wastewater, building or machinesurface), or an edible substrance or fluid (e.g., food, water). In someembodiments where the fluid is blood, after removal of themicrobe/pathogen from the blood collected from a subject with themicrobe-targeting magnetic microbeads, the blood can be circulated backto the same subject as a therapeutic intervention. In some embodiments,the microbe-targeting magnetic microbeads can be used in diagnostics asa means of collecting potential pathogens for identification; not onlyin the diagnosis of disease, but in the identification of water- orfood-borne pathogens, particulates or other contaminants. Alternatively,the solid substrate can comprise a hollow-fiber reactor or any otherblood filtration membrane or flow device (e.g., a simple dialysis tube,spiral mixer or static mixer) or other resins, fibers, or sheets toselective bind and sequester the biological pathogens.

The magnetic microbeads can be of any shape, including but not limitedto spherical, rod, elliptical, cylindrical, and disc. In someembodiments, magnetic beads having a substantially spherical shape anddefined surface chemistry can be used to minimize chemical agglutinationand non-specific binding. As used interchangeably herein, the terms“magnetic microbeads” and “magnetic beads” can refer to a nano- ormicro-scale particle that is attracted or repelled by a magnetic fieldgradient or has a non-zero magnetic susceptibility. The magneticmicrobeads can be ferromagnetic, paramagnetic or super-paramagnetic. Insome embodiments, magnetic microbeads can be super-paramagnetic. In someembodiments, magnetic microbeads can have a polymer shell for protectingthe microbe-targeting molecule from exposure to iron provided that thepolymer shell has no adverse effect on the magnetic property. Forexample, biocompatible polymer-coated magnetic microbeads can be used toremove microbes/pathogens from a test sample, e.g., a biological fluid,such as blood.

The magnetic microbeads can range in size from 1 nm to 1 mm. Forexample, magnetic microbeads can be about 2.5 nm to about 500 μm, orabout 5 nm to about 250 μm in size. In some embodiments, magneticmicrobeads can be about 5 nm to about 100 μm in size. In someembodiments, magnetic microbeads can be about 0.01 μm to about 10 μm insize. In some embodiments, magnetic microbeads can be about 0.05 μm toabout 5 μm in size. In some embodiments, magnetic microbeads can beabout 0.08 μm to about 1 μm in size. In one embodiment, magneticmicrobeads can be about 10 nm to about 10 μm in size. In someembodiments, the magnetic microbeads can be magnetic nanobeads, e.g.,with a size ranging from about 1 nm to about 1000 nm, from about 10 nmto about 500 nm, from about 25 nm to about 300 nm, from about 40 nm toabout 250 nm, or from about 50 nm to about 200 nm. In one embodiment,the magnetic microbeads can be magnetic nanobeads with a size of about50 nm to about 200 nm. Magnetic microbeads can be manipulated usingmagnetic field or magnetic field gradient. Such particles commonlyconsist of magnetic elements such as iron, nickel and cobalt and theiroxide compounds. Magnetic microbeads are well-known and methods fortheir preparation have been described in the art. See, e.g., U.S. Pat.No. 6,878,445; U.S. Pat. No. 5,543,158; U.S. Pat. No. 5,578,325; U.S.Pat. No. 6,676,729; U.S. Pat. No. 6,045,925; and U.S. Pat. No.7,462,446; and U.S. Patent Publications No. 2005/0025971; No.2005/0200438; No. 2005/0201941; No. 2005/0271745; No. 2006/0228551; No.2006/0233712; No. 2007/01666232; and No. 2007/0264199, the contents ofwhich are incorporated herein by reference.

Magnetic microbeads are also widely and commercially available, with orwithout functional groups capable of binding to coupling molecules.Magnetic microbeads functionalized with various functional groups, e.g.,amino groups, carboxylic acid groups, epoxy groups, tosyl groups, orsilica-like groups, are also widely and commercially available. Suitablemagnetic microbeads are commercially available such as from AdemTech,Miltenyi, PerSeptive Diagnostics, Inc. (Cambridge, Mass.); InvitrogenCorp. (Carlsbad, Calif.); Cortex Biochem Inc. (San Leandro, Calif.); andBangs Laboratories (Fishers, Ind.). In particular embodiments, magneticmicrobeads that can be used herein can be any DYNABEADS® magneticmicrobeads (Invitrogen Inc.), depending on the substrate surfacechemistry.

Microbe-Targeting Cells:

In some embodiments, the substrate to which the engineeredmicrobe-targeting molecule binds can be a living cell, or a biologicaltissue or organ. For example, the living cells can be associated with animmune response, and such cells include, but are not limited to, aphagocyte (macrophage, neutrophil, and dendritic cell), mast cell,eosinophil, basophil, and/or natural killer cell. Alternatively, theliving cell can be the cell of biological tissues or organs of theimmune system, such as spleen, lymph nodes, lymphatic vessels, tonsils,thymus, bone marrow, Peyer's patches, connective tissues, mucousmembranes, the reticuloendothelial system, etc. In some embodiments, thesurface to which the engineered microbe-targeting molecules bind canalso be the extracellular matrix of one or more of these tissues ororgans.

Microbe-Binding Microtiter Plates:

In some embodiments, the bottom surface of microtiter wells can becoated with the engineered microbe-targeting molecules described herein,e.g., for detecting and/or determining the amount of microbes in asample. After microbes or pathogens in the sample binding to theengineered microbe-targeting molecules bound to the microwell surface,the rest of the sample can be removed. Detectable molecules that canalso bind to microbes or pathogens (e.g., an engineeredmicrobe-targeting molecule conjugated to a detectable molecule asdescribed herein) can then be added to the microwells withmicrobes/pathogens for detection of microbes/pathogens. Various signaldetection methods for determining the amount of proteins, e.g., usingenzyme-linked immunosorbent assay (ELISA), with different detectablemolecules have been well established in the art, and those signaldetection methods can also be employed herein to facilitate detection ofthe signal induced by microbes/pathogens binding on the engineeredmicrobe-targeting molecules.

Microbe-Binding Dipsticks/Test Strips:

In some embodiments, the engineered microbe-targeting molecules can beadapted for use in a dipstick and/or a test strip for detection ofmicrobes or pathogens. For example, a dipstick and/or a test strip caninclude at least one test area containing one or more engineeredmicrobe-targeting molecules described herein. In some embodiments, theengineered microbe-targeting molecules can be conjugated or attached toa test area surface of the dipstick and/or a test strip. Methods forconjugating a protein to a substrate surface are known in the art,including, but not limited to direct cross-linking, indirectcross-linking via a coupling agent (e.g., a functional group, a peptide,a nucleic acid matrix such as DNA matrix), absorption, or any otherart-recognized methods known in the art.

In one embodiment, about 1 μg to about 100 μg microbe-binding moleculescan be coated on or attached to a dipstick or membrane surface. Inanother embodiment, about 3 μg to about 60 μg microbe-binding moleculescan be coated on or attached to a dipstick or membrane surface. In someembodiments, about 0.1 mg/mL to about 50 mg/mL, about 0.5 mg/mL to about40 mg/mL, about 1 mg/mL to about 30 mg/mL, about 5 mg/mL to about 20mg/mL microbe-binding molecules can be coated on or attached to adipstick or membrane surface. In one embodiment, about 11.5 mg/mLmicrobe-binding molecules can be coated on or attached to a dipstick ormembrane surface.

In some embodiments, the engineered microbe-targeting molecule(s)conjugated to the dipstick and/or a test strip can further comprise adetectable label as described herein. In one embodiment, the detectablelabel can include a microbial enzyme substrate conjugated to adetectable moiety. Such detectable moiety is undetectable whenconjugated to the microbial enzyme substrate, but becomes a detectableentity (e.g., a light-emitting signal) in the presence of an enzymepossessed or secreted by the microbe. See, e.g., WO 2011/103144, for theuse of such detectable label in detection of microbes, the content ofwhich is incorporated herein by reference.

In some embodiments, the dipstick and/or a test strip can furthercomprise at least one reference area or control area for comparison witha readout signal determined from the test area. The reference areagenerally excludes the engineered microbe-targeting molecules, e.g., toaccount for any background signal. In some embodiments, the referencearea can include one or more known amounts of the detectable label thatthe engineered microbe-targeting molecules in the test area encompass.In such embodiments, the reference area can be used for calibration suchthat the amount of microbes in a test sample can be estimated orquantified.

The dipstick and/or a test strip can be in any shape and/or in anyformat, e.g., a planar shape such as a rectangular strip or a circulardisk, or a curved surface such as a stick. Alternatively, a continuousroll can be utilized, rather than discrete test strips, on which thetest area(s) and optionally reference area(s) are present in the form ofcontinuous lines or a series of spots.

The dipstick and/or a test strip can be made of any material, including,without limitations, paper, nitrocellulose, glass, plastic, polymer,membrane material, nylon, and any combinations thereof. In oneembodiment, the dipstick and/or a test strip can include paper. In oneembodiment, the dipstick and/or a test strip can include nylon.

The microbe-binding dipsticks and/or test strips described herein can beused as point-of-care diagnostic tools for microbe or pathogendetection. By way of example only, a microbe-binding dipstick or teststrip (e.g., made of membrane material such as nylon) can be broughtinto contact with a test sample (e.g., a blood sample) from a patient ora subject, and incubated for a period of time, e.g., at least about 15seconds, at least about 30 seconds, at least about 1 min, at least about2 mins, at least about 5 mins, at least about 10 mins, at least about 15mins, at least about 30 mins, at least about 1 hour or more. In someembodiments, the incubated dipstick or test strip can then be incubatedin a blocking agent (e.g., BSA, normal serum, casesin, non-fat dry milk,and/or any commercially-available blocking agents to minimizenon-specific binding). Depending on different embodiments of theengineered microbe-targeting molecules, in some embodiments, themicrobe-binding dipstick or test strip after contact with a test sample(e.g., a blood sample) can be further contacted with at least oneadditional agent to facilitate detection of pathogen, and/or to increasespecificity of the pathogen detection. For example, some embodiments ofthe dipstick or test strip after contact with a test sample (e.g., ablood sample) can be further contacted with a detectable label that isconjugated to a molecule that binds to a microbe and/or microbialmatter. Examples of such molecules can include, but are not limited to,one or more embodiments of the engineered microbe-targeting moleculedescribed herein, an antibody specific for the microbes or pathogens tobe detected, a protein, a peptide, a carbohydrate or a nucleic acid thatis recognized by the microbes or pathogens to be detected, and anycombinations thereof.

In some embodiments, the readout of the microbe-binding dipsticks and/ortest strips can be performed in a system or device, e.g., a portabledevice. The system or device can display a signal indicating thepresence or the absence of a microbial infection in a test sample,and/or the extent of the microbial infection.

Generally, the diagnosis of infection relies on indirect or directevidence. The indirect evidence relies on the detection of an adaptedand specific host response directed against the pathogen. The directevidence relies on the culture of the microorganism from the infectedsite, amplification and detection of pathogen-specific nucleic acids orthe detection of a specific antigen in blood or urine; however, existingtechnologies only allow detection of living pathogens and not non-livingmicrobial matter, such as endotoxins, that can have devastating effectson patient survival.

Specific antigen detection is widely used for a variety of infectiousdiseases, most commonly for legionellosis (Legionella pneumophilaserotype 1 in urine), malaria (Plasmodium falciparum in blood) and withless success with Streptococcus pneumonia infection (in urine). However,direct antigen detection can only be used to rule in or rule out aspecific etiology and cannot identify most bacteria.

As described herein, engineered microbe-binding molecules or substrates(e.g., FcMBL molecules or FcMBL-bound magnetic microbeads) can bind tothe surface of a wide array of microbes including pathogens, e.g., butnot limited to, bacterial, fungal, parasitic or viral. For example, insome embodiments, blood or urine or any other biological fluid can besubjected to microbial capture by the engineered microbe-bindingmolecules or substrates (e.g., FcMBL molecules or FcMBL-bound magneticmicrobeads) and adequate controls (e.g., non-specific binding control bynon-relevant protein coated magnetic microbeads). Accordingly,engineered microbe-binding molecules or substrates (e.g., FcMBL orFcMBL-coated magnetic microbeads) can be used to bind microbes such asbacteria for diagnostic or therapeutic applications.

Not only can the engineered microbe-binding molecules or substrates bindto at least a portion of a cell surface of a microbe, the engineeredmicrobe-binding molecules or substrates can also capture microbialmatter (e.g., microbe-originating cell fragments or matter derived frommicrobes circulating in biological fluids including endotoxins, e.g.,during the course of an infection, even in the absence of bacteremia, orfound on an environmental surface, food or water, a pharmaceuticalproduct or a medical device). The presence of such microbial cellfragments or microbe-derived matter can be used, alone or in combinationwith detection of an intact microbe, for diagnostic applications, e.g.,the presence of pathogen-originating cell fragments or matter derivedfrom pathogens can be diagnostic of an infectious disease in a subject,or a microbial contamination on an environmental surface, food or water,a pharmaceutical product, or a medical device. Moreover, thebiochemical/proteomic (MALDI-TOF, multiple mass spectrometry (e.g., MSn)or specific antibody or aptamer based) analysis of the bound products(e.g., microbial matter or microbes bound onto an engineeredmicrobe-binding molecule or substrate) can allow recognition of elementspathognomonic for microbes.

Accordingly, provided herein also include methods for detection of thepresence or absence of a microbe and/or microbial matter in an organ, atissue, and/or a cell in a subject (including blood, normally sterilefluids or virtual cavities). For example, the presence or absence of amicrobe and/or microbial matter can be detected by capture of a microbeand/or non-viable microbial matter or particles circulating in thesubject's body fluid, e.g., blood, or found in other fluids such asurine, or in any other organ sampled by any appropriate means (e.g., butnot limited to, biopsy, puncture, aspiration, and lavage).

The inventors have discovered that, in some embodiment, FcMBL capturednot only whole bacteria for concentration and direct analysis but alsonon-viable microbial matter. Such binding can be quantified by a microbebinding assay based on the capture of this microbial matter on theengineered microbe-binding molecules or substrates (e.g., FcMBL-coatedmicrobeads). The detection of this material can be performed usingenzyme-linked engineered microbe-binding molecules described herein(e.g., FcMBL) or fluorescent-linked engineered microbe-binding moleculesdescribed herein (e.g., FcMBL). The engineered microbe-binding molecules(e.g., FcMBL) can be multimerized on the surface of a desired substrate(e.g., a magnetic bead) to form a microbe-binding substrate for enhancedavidity. Examples 16-17 show that engineered microbe-binding moleculesdescribed herein (e.g., FcMBL) can detect live and dead microbes as wellas microbial matter (including, but not limited to, fragments of amicrobe and endotoxins) in a biological sample (e.g., blood sample), andthe detection results correlate with clinical symptoms or morbidity ofan infection.

Accordingly, in some embodiments, the presence of intact microbes and/ormicrobial matter (including microbe cell fragments or matter derivedfrom a microbe) bound on the engineered microbe-binding molecules orsubstrates can be used as a marker for infection or contamination.Current generic biomarkers for infection include molecules, for example,cytokines; acute phase proteins such as CRP, procalcitonin, andfibrinogen; erythrocyte sedimentation rate (ESR), and elevated ordiminished leukocyte counts. However, these generic biomarkers are notspecific to infection, but are also involved in non-infectiousinflammation.

In contrast, binding of microbes or fragments thereof (including matterderived from microbes) on an engineered microbe-binding molecule and/orsubstrate can not only be used for infection of a sampled organ ortissue or cell(s) (blood or otherwise) but also to any major infectiousprocess ongoing anywhere in the body where sufficient microbialdestruction or catabolism results in the presence of microbial matter inthe bloodstream, urine or any other conveniently accessed fluid. Thereis currently no biological marker for infection that does notcross-react with generic non-infectious inflammation. Thus, this is amajor breakthrough in the management of patients suspected of infection.Without wishing to be bound, not only can the engineered microbe-bindingmolecules and/or substrates be used to detect an infection in a subject(e.g., a mammalian subject), but they can also be used to detect thepresence or absence of a microbe in any environment or on any devicewhere a microbe can be present, including but are not limited to,biomedical devices, clinics or hospitals, ponds or water reservoirs,wastewater, water farms (including hydroponics), and/or food processingplants or machines.

Indeed, the inventors have collected blood from de-identified,hospitalized patients and demonstrated, in some embodiments, that theFcMBL assay is positive in patients with negative blood cultures andcorrelates strongly with the diagnosis of infection. Thus, in someembodiments, the FcMBL assay is more sensitive than conventional bloodcultures for detection of an infection. In some embodiments, the FcMBLassay can be used for early diagnosis of an infection. In someembodiments, the engineered microbe-binding molecules and/or substratesand/or diagnosis/detection processes described herein can detectpresence of a microbe and/or microbial matter in a test sample whichpreviously yielded a negative result in a traditional diagnosis method(e.g., a blood culture). Accordingly, the engineered microbe-bindingmolecules and/or substrates and/or diagnosis/detection process describedherein can enable a more sensitive and faster diagnosis than thetraditional diagnostic method (e.g., a blood culture).

Further, in some embodiments, the wide spectrum of the engineeredmicrobe-binding molecules or substrates (e.g., FcMBL molecules orFcMBL-coated magnetic microbeads) can enable the capture of mostclinically relevant bacterial species. The presence of microbial matteror fragments of microbes can reflect deep tissue infection as theygenerally find its way into the bloodstream and most likely the urine.The capture and characterization of this microbial matter or fragmentsof microbes can be used as evidence markers specific for a givenmicrobial species, thus allowing the diagnosis and/or identification ofa microbe causing infection anywhere in an organism.

For example, the use of one or more specific antibodies can allowcharacterization of the nature and/or types of the microbial materialbound to the engineered microbe-binding molecules or substrates.Specific detection of certain molecules (e.g., proteins, carbohydrates,lipids) present on a microbe surface, such as Lipid A on E. coli or anyother molecules on a microbe of interest, can allow furtherdiscrimination of samples or identification of microbes present in thesamples. Without wishing to be limiting, as shown in Example 16 and FIG.33, in order to determine if the captured microbes and/or fragmentsthereof were associated with E. coli, FcMBL-coated magnetic beads withcaptured microbes and/or fragments thereof can be further contacted witha specific antibody raised against Escherichia coli lipopolysaccharideLipid A (anti-LPS Lipid A antibody). As shown in the bottom panel ofFIG. 33, the microbes and/or fragments thereof captured on theFcMBL-coated magnetic beads did not bind to anti-LPS antibodies,indicating that the microbes and/or microbial fragments bound to theFcMBL-coated microbeads were unlikely associated with E. coli. Incontrast, the microbes and/or fragments thereof captured on theFcMBL-coated magnetic microbeads bound to anti-LPS antibodies,indicating that the microbes and/or microbial fragments bound to theFcMBL-coated microbeads were likely associated with E. coli.Accordingly, the screening of a library of antibodies directed against aplurality of microbes (including pathogens) can allow direct diagnosisof microbe-specific infections, e.g., anywhere in the body of a subjectby a simple blood or urine test available in less than three hours inany microbiology laboratory equipped for magnetic separation.

In a different embodiment, a rapid test can be performed using a“dipstick” format. For example, a membrane spotted with lines ofmicrobial species-specific antibodies (instead of FcMBL molecules asshown in FIG. 13) can be incubated with the microbe-binding substrates(e.g., FcMBL-coated microbeads) previously incubated with a fluid testsample. The microbe-binding substrates (e.g., FcMBL-coated microbeads)captured by proper antibodies on the membrane can form a detectable band(e.g., rust-colored for FcMBL-coated magnetic microbeads) on themembrane, indicating the species (one or many) of which microbial matteror microbes was captured.

Other than antibody-based characterization methods, other known methodssuch as mass spectrometric characterization methods or PCR analysis canalso be used to characterize and/or identify the species of a microbecaptured on the engineered microbe-binding molecules and/or substrates.In some embodiments, the microbe-binding molecules and/or substrateswith captured microbes and/or microbial matter/fragments can be washedprior to any further characterization methods such as mass spectrometriccharacterization methods.

In some embodiments, the engineered microbe-binding molecules and/orsubstrates with captured microbes and/or microbial matter/fragments canbe subjected to direct analysis for characterization and/oridentification of species of microbes and/or microbial matter boundthereon. For example, the engineered microbe-binding molecules and/orsubstrates with captured microbial materials can be directly subjectedto MALDI-TOF analysis (e.g., without separation of the capturedmicrobial materials from the engineered microbe-binding molecules and/orsubstrates).

Alternatively, any art-recognized protocols or methods described hereincan be applied on the engineered microbe-binding molecules and/orsubstrates to isolate bound microbes and/or microbialcompounds/fragments from the engineered microbe-binding molecules and/orsubstrates prior to any characterization analysis. Exemplary methods torecover or isolate bound microbes and/or microbial compounds/fragmentsfrom the engineered microbe-binding molecules and/or substrates, priorto any characterization analysis, include, but are not limited to, Ca²⁺chelation to release captured materials from the engineeredmicrobe-binding molecules and/or substrates; lowering pH to releasebinding mediated by Fc-protein A interaction; protein extraction usingformic acid and acetonitrile, and any combinations thereof. The controlmicrobeads (e.g., microbeads coated with molecules that do not react tomicrobes) can be treated similarly for baseline determination.

In some embodiments, the extracted captured material from the engineeredmicrobe-binding molecules and/or substrates and/or non-specificcontrol-bound material can be subjected to PCR analysis. For example,the identity of the extracted captured material can be determined bydetecting the presence or absence of a gene encoding a protein specificto a microbe species. Thus, the presence of one or more microbespecies-specific genes (s) can be indicative of the correspondingmicrobe species bound on the engineered microbe-binding molecules.

In some embodiments, extracted captured material from the engineeredmicrobe-binding molecules and/or substrates, and/or non-specificcontrol-bound material can be subjected to mass spectrometric analysis,including but not limited to, MALDI-TOF or MALDI-TOF-TOF. Thenon-specific control-bound material can establish a baseline for thecomposition of the medium tested. This profile can be used as referencefor the analysis of the material bound to the engineered microbe-bindingmolecules and/or substrates. Peaks present in the control-bound samplescan be subtracted from the profile obtained from the material bound tothe engineered microbe-binding molecules and/or substrates. The specificprofile of the material that was bound to the microbe-binding moleculesand/or substrates (e.g., after subtraction of the reference profile) canconstitute a microbe/microbial fragment signature. Both positive and/ornegative charge analysis can be performed to identify informative peaks.

Recognition of a microbe signature can be analyzed by any known methodsin the art. For example, a microbe/microbial fragment signature can berecognized by comparing the specific profile of the material that wasbound to the microbe-binding molecules and/or substrates to one or moremicrobe/microbial fragment signature libraries, e.g., using matchingcomparison algorithms based on the previously accumulated profiles.

For identification of microbe species, depending on origins of microbes,a microbe/microbial fragment signature library can be established by invivo or in situ samples such as clinical-trial derived samples and/orenvironment derived samples (e.g., samples collected from a clinicalsetting, culture medium, food processing plant, water source). Forexample, blood (or other biological fluids) of patients infected withknown microbes, e.g., pathogens, can be analyzed and a microbialmaterial signature can be characterized. Recognition of the signature inthe same clinical context can establish the family/genus/speciesdiagnosis.

Additionally or alternatively, another microbe/microbial fragmentlibrary can be established from in vitro analysis of microbes' bindingmoieties to engineered microbe-binding molecule(s) described herein,wherein the microbes can be subjected to mechanical or chemical orantibiotic lysis or autolysis. The microbial material can be captured indifferent media, buffer, urine, blood or any appropriate medium.

The diagnostic profiles can be matched to any reference profiles, e.g.,specific in vivo or in situ derived microbe profiles and/or specificin-vitro derived microbes profiles for identification with a probabilityscore for generic infection, clades level, family level, genus level orspecies level identification.

Further, methods for detection of the presence or absence of a microbeand/or microbial matter on an environmental surface, food or water, apharmaceutical product, or a medical device by capture of a microbeand/or non-viable microbial matter or particles present thereon are alsowithin the scope described herein. In some embodiments, the methods ofany aspect described herein can be used to screen pharmaceuticalproducts (e.g., drugs, therapeutic agents or imaging agents), and/ormedical devices (e.g., fluid delivery devices, or implantable devices)for the presence or absence of a microbe and/or microbial matter(including but not limited to endotoxin produced by a microbe, e.g., agram-negative microbe such as E. coli and/or a gram-positive microbesuch as S. aureus). In one embodiment, the method can be used to screenpharmaceutical products (e.g., drugs, therapeutic agents or imagingagents), and/or medical devices (e.g., fluid delivery devices, orimplantable devices) for the presence or absence of endotoxin producedby a microbe, e.g., a gram-negative microbe such as E. coli and/or agram-positive microbe such as S. aureus.

Exemplary Optimization or Modifications of Microbe-Targeting Substrates

In accordance with at least some embodiments described herein,engineered microbe-binding molecules and/or substrates (e.g.,FcMBL-bound paramagnetic microbeads) can bind to a surface of a varietyof microbes and/or microbial matter described herein, e.g., but notlimited to, bacterial, fungal, parasitic or viral. In some embodiments,a number of factors such as the orientation of engineeredmicrobe-binding molecules (e.g., FcMBL) conjugated to or coated on asubstrate (e.g., a paramagnetic microbead), size of a substrate (e.g., amicrobead), selection of linkers and microbe surface-binding domainsused in constructing an engineered microbe-targeting molecule, microbialassay condition, and any combinations thereof, can be optimized forbinding of the microbe-targeting substrates to microbes.

Optimization of Substrate Size and Densities of EngineeredMicrobe-Targeting Molecules on the Substrate:

Additionally or alternatively, the density of engineered microbe-bindingmolecules (e.g., FcMBL) conjugated to or coated on a substrate (e.g., amicrobead) can be optimized to capture microbes.

In some embodiments where the engineered microbe-binding molecule isFcMBL, the FcMBL differs from recombinant wild-type MBL in that theFcMBL is a dimeric protein with two Carbohydrate Recognition Domain(CRD) heads whereas wild-type MBL has 9-18 heads in groups of 3. Theaffinity of the individual heads is 10⁻³ and MBL binding to microbesurfaces requires binding of multiple CRD heads to give high aviditybinding. In order to achieve this high avidity with the dimeric FcMBLprotein, in some embodiments, a plurality of (e.g., at least about 2, atleast about 5, at least about 10, at least about 25, at least about 50,at least about 100, at least about 1000, at least about 10⁴, at leastabout 10⁵, at least about 10⁶, at least about 10⁷) engineeredmicrobe-binding molecules (e.g., FcMBL) can be multiplexed on a surfaceof a substrate (e.g. magnetic microbeads such as the MYONE™ Streptavidinmicrobeads from Life Technologies). The number of the engineeredmicrobe-binding molecules conjugated to a substrate can vary withavailable surface area of a substrate.

Accordingly, a number of factors, including density of engineeredmicrobe-binding molecules on a substrate, size of the substrate, and/orsize of the engineered microbe-binding molecules, can be varied tooptimize binding of microbes to the engineered microbe-bindingsubstrates (e.g., but not limited to, FcMBL-coated beads). Someexemplary optimizations/modifications can include, but are not limitedto, using a substrate (e.g., but not limited to, a microbead) ofdifferent sizes; varying the density of engineered microbe-bindingmolecules (e.g., but not limited to, FcMBL) on the substrate (e.g., butnot limited to, a microbead) by binding the engineered microbe-bindingmolecules (e.g., but not limited to, FcMBL) to a substrate scaffold invarious oriented arrays, e.g., but not limited to DNA, aptamers, orextracellular matrix (e.g., fibronectin); producing fusion proteins ofmicrobe-binding domain(s) (e.g., but not limited to, MBL CRD head andneck regions) bound to a linker described herein or fusion partner (orlinker described herein) of different sizes (e.g., between about 100 kDato about 1000 kDa or between about 250 kDa to about 750 kDa. Anexemplary fusion partner can include, but is not limited to, the Fcportion of IgM, which is about 500 kDa); producing fusion proteins ofmicrobe-binding domain(s) (e.g., but not limited to, MBL CRD head andneck regions) with multimeric (e.g., at least dimeric, at leasttrimeric) linkers described herein or fusion partners (or linkersdescribed herein); and any combinations thereof. As used herein, theterm “multimeric linker” or “multimeric fusion partner” refers to alinker or fusion partner comprising two or more identical linker unitsfor providing attachment of microbe-binding domains. By way of exampleonly, a trimeric linker or fusion partner is a linker or fusion partnercomprising three identical linker units for attachment ofmicrobe-binding domains.

The binding of any microbe to a microbe-binding substrate describedherein can be determined by any methods known in the art and/ordescribed herein, such as by ELISA-colorimetric assay or antibody-basedimaging methods described in the Examples. Accordingly, themicrobe-binding substrate can be optimized for detection of a microbe,e.g., by varying its density and/or size of engineered microbe-bindingmolecules, its substrate structure and/or size, and then determiningtheir effects on the binding of the microbe to the microbe-bindingsubstrate.

For example, Example 18 shows an exemplary method to evaluate themicrobe-capture efficiency of microbe-targeting magnetic microbeads(e.g., FcMBL-coated magnetic microbeads) having different sizes. In someembodiments, a microbead (e.g., a magnetic microbead or a non-magneticmicrobead) as a substrate for attachment of engineered microbe-bindingmolecules can have a size of about 10 nm to 10 μm, about 20 nm to about5 μm, about 40 nm to about 1 μm, about 50 nm to about 500 nm, or about50 nm to about 200 nm. Without wishing to be bound by theory, the sizeof a microbead can be smaller than the size of a microbe so that morethan one microbead (e.g., at least 2, at least 3, at least 4, at least5, at least 10 or more) can bind to the same microbe for enhancedcapture and increased detection sensitivity.

Additionally, the density of the engineered microbe-binding molecules ona surface of the microbe-targeting substrate can be optimized formicrobial binding. In order to enhance binding of a specific microbe tothe microbe-targeting substrate, the distance between any twomicrobe-binding molecules on a surface of the microbe-targetingsubstrate can be less than the size of a microbe. Therefore, a microbecan bind to more than one microbe-binding molecules (e.g., at least 2,at least 3, at least 4, at least 5, at least 6, at least 7, at least 8,at least 9, at least 10, or more) present on the microbe-targetingsubstrate with a greater combined binding strength.

In some embodiments, the microbe-targeting substrates can comprise ontheir surfaces a saturating amount of the engineered microbe-bindingmolecules described herein. As used herein, the term “saturating amount”refers to the maximum number or amount of engineered microbe-bindingmolecules that can be conjugated to and/or coated on a surface of asubstrate. The saturating amount of the engineered microbe-bindingmolecules that can be present on a surface of a substrate is dependenton a number of factors such as size and/or structure of the engineeredmicrobe-binding molecules, size and/or structure of the substrate,orientation of the engineered microbe-binding molecules present on thesubstrate, and any combinations thereof.

Selection of Linkers and Microbe Surface-Binding Domain Used inConstructing an Engineered Microbe-Targeting Molecule and MicrobialAssay Condition:

In some embodiments, a linker can be selected to provide binding sitesof a microbe, wherein the binding interaction of the microbe to thelinker is different from the interaction of the microbe to the microbesurface-binding domain. For example, in an engineered microbe-bindingmolecule where the linker is a Fc molecule and the microbesurface-binding domain is derived from MBL or a fragment thereof, the Fclinker allows protein A or protein G binding, which iscalcium-independent, while the MBL binding domain requires calcium ionsfor binding with a microbe. Accordingly, a protein A-expressing (e.g.,S. aureus) or protein G-expressing microbe can bind to both MBL bindingdomain and Fc linker in the presence of calcium ions, but can bind toonly Fc linker in the absence of calcium ions. In contrast, a protein A-and protein G-negative microbe (e.g., E. coli) generally binds toneither MBL binding domain nor Fc linker in the absence of calcium ions.In such embodiments, by controlling the amount of calcium ions presentin a microbial assay, one can distinguish protein A- or proteinG-expressing microbes (e.g., S. aureus) from protein A- and proteinG-negative microbes (e.g., E. coli). Additional details of suchembodiments can be found in later sections “Exemplary Process forCapture and/or Detection of a Microbe and/or Microbial Matter in a TestSample” and “Exemplary Embodiments of Methods for Diagnosing a MicrobialInfection.”

Exemplary Process for Capture and/or Detection of a Microbe and/orMicrobial Matter in a Test Sample

In one aspect, a process for detecting a microbe and/or microbial matterin a test sample is described herein. As shown in FIG. 17, the process1200 comprises the optional step 1202 (preprocessing of the sample),step 1204 (processing of the sample), step 1206 comprising 1208 (microbecapture) and 1210 (microbe separation), and 1212 (microbe detection).While these are discussed as discrete processes, one or more of thepreprocessing, processing, capture, microbe separation, and detectioncan be performed in a microfluidic device. Use of a microfluidic devicecan automate the analysis process and/or allow analysis of multiplesamples at the same time. One of skill in the art is well aware ofmethods in the art for collecting, handling and processing biologicalfluids which can be used in the practice of the present disclosure. Theprocess described herein can allow sample analysis at in short timeperiods. For example, the process can be completed in less than 6 hours,less than 5 hours, less than 4 hours, less than 3 hours, less than 2hours, less than 1 hour, less than 30 minutes. In some embodiments,presence and identity of a microbe in the sample can be done within 10minutes to 60 minutes of starting the process.

In some embodiments, the sample can be a biological fluid, e.g., blood,plasma, serum, lactation products, amniotic fluids, sputum, saliva,urine, semen, cerebrospinal fluid, bronchial aspirate, perspiration,mucus, liquefied stool sample, synovial fluid, lymphatic fluid, tears,tracheal aspirate, and any mixtures thereof. For example, the sample canbe a whole blood sample obtained from a subject.

The process described herein can be utilized to detect the presence of amicrobe in a sample of any given volume. In some embodiments, samplevolume is about 0.25 ml to about 50 ml, about 0.5 ml to about 25 ml,about 1 ml to about 15 ml, about 2 ml to about 10 ml. In someembodiments, sample volume is about 5 ml. In one embodiment, samplevolume is 8 ml.

1202 (Sample Preprocessing):

It can be necessary or desired that a test sample, such as whole blood,be preprocessed prior to microbe detection as described herein, e.g.,with a preprocessing reagent. Even in cases where pretreatment is notnecessary, preprocessing can be optionally done for mere convenience(e.g., as part of a regimen on a commercial platform). A preprocessingreagent can be any reagent appropriate for use with the assays orprocesses described herein.

The sample preprocessing step generally comprises adding one or morereagent to the sample. This preprocessing can serve a number ofdifferent purposes, including, but not limited to, hemolyzing bloodcells, dilution of sample, etc. The preprocessing reagents can bepresent in the sample container before sample is added to the samplecontainer or the preprocessing reagents can be added to a sample alreadypresent in the sample container. When the sample is a biological fluid,the sample container can be a VACUTAINER®, e.g., a heparinizedVACUTAINER®.

The preprocessing reagents include, but are not limited to, surfactantsand detergents, salts, cell lysing reagents, anticoagulants, degradativeenzymes (e.g., proteases, lipases, nucleases, lipase, collagenase,cellulases, amylases and the like), and solvents, such as buffersolutions.

In some embodiments, a preprocessing reagent is a surfactant or adetergent. In one embodiment, the preprocessing reagent is Triton X100.

Amount of preprocessing reagent to be added can depend on a number offactors. Generally, the preprocessing reagent is added to a finalconcentration of about 0.1 mM to about 10 mM. If a liquid, thepreprocessing reagent can be added so as to dilute the sample at least10%, at least 20%, at least 30%, at least 40%, at least 50%, at least60%, at least 60%, at least 80%, at least 90%, at least 1-fold, at least2-fold, at least 3-fold, or at least 5-fold.

After addition of the preprocessing reagent, the reagent can be mixedinto the sample. This can be simply accomplished by agitating thesample, e.g., shaking or vortexing the sample and/or moving the samplearound, if it is in a microfluidic device.

After addition of the preprocessing reagent, the sample mixture can beincubated for a period of time, e.g., for at least one minute, at leasttwo minutes, at least three minutes, at least four minutes, at leastfive minutes, at least ten minutes, at least fifteen minutes, at leastthirty minutes, at least forty-five minutes, or at least one hour. Suchincubation can be at any appropriate temperature, e.g., room-temperature(e.g., about 16° C. to about 30° C.), a cold temperature (e.g. about 0°C. to about 16° C.), or an elevated temperature (e.g., about 30° C. toabout 95° C.). In some embodiments, the sample is incubated for aboutfifteen minutes at room temperature. In some embodiments, incubation isfor about 5 seconds to about 60 seconds. In some embodiments, there isno incubation and the sample mixture is used directly in the sampleprocessing step.

1204 (Sample Processing):

After the optional preprocessing step, the sample can be optionallyprocessed by adding one or more processing reagents to the sample. Theseprocessing reagents can serve to lyse cells, degrade unwanted moleculespresent in the sample and/or dilute sample for further processing. Theseprocessing reagents include, but are not limited to, surfactants anddetergents, salts, cell lysing reagents, anticoagulants, degradativeenzymes (e.g., proteases, lipases, nucleases, lipase, collagenase,cellulases, amylases and the like), and solvents, such as buffersolutions. Amount of the processing reagent to be added can depend onthe particular sample to be analyzed, the time required for the sampleanalysis, identity of the microbe to be detected or the amount ofmicrobe present in the sample to be analyzed.

It is not necessary, but if one or more reagents are to be added theycan present in a mixture (e.g., in a solution, “processing buffer”) inthe appropriate concentrations. Amount of the various components of theprocessing buffer can vary depending upon the sample, microbe to bedetected, concentration of the microbe in the sample, or time limitationfor analysis.

Generally, addition of the processing buffer can increase the volume ofthe sample by 5%, 10%, 15%, 20% or more. In some embodiments, about 50μl to about 5000 μl of the processing buffer are added for each ml ofthe sample. In some embodiments, about 100 μl to about 250 μl of theprocessing buffer are added for each ml of the sample. In oneembodiment, about 800 μl of the processing buffer are added for each 200μl of the sample.

In some embodiments, a detergent or surfactant comprises about 5% toabout 20% of the processing buffer volume. In some embodiment, adetergent or surfactant comprises about 5% to about 15% of theprocessing buffer volume. In one embodiment, a detergent or surfactantcomprises about 10% of the processing buffer volume.

Exemplary surfactants and detergents include, but are not limited to,sulfates, such as, ammonium lauryl sulfate, sodium dodecyl sulfate(SDS), and sodium lauryl ether sulfate (SLES) sodium myreth sulfate;sulfonates, such as, dioctyl sodium sulfosuccinate (Docusates),perfluorooctanesulfonate (PFOS), perfluorobutanesulfonate, alkyl benzenesulfonates, and3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS);3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate(CHAPSO); phosphates, such as alkyl aryl ether phosphate and alkyl etherphosphate; carboxylates, such as fatty acid salts, sodium stearate,sodium lauroyl sarcosinate, perfluorononanoate, and perfluorooctanoate(PFOA or PFO); octenidine dihydrochloride; alkyltrimethylammonium salts,such as cetyl trimethylammonium bromide (CTAB) and cetyltrimethylammonium chloride (CTAC); cetylpyridinium chloride (CPC);polyethoxylated tallow amine (POEA); benzalkonium chloride (BAC);benzethonium chloride (BZT); 5-Bromo-5-nitro-1,3-dioxane;dimethyldioctadecylammonium chloride; dioctadecyldimethylammoniumbromide (DODAB); sultaines, such as cocamidopropyl hydroxysultaine;cetyl alcohol; stearyl alcohol; cetostearyl alcohol (consistingpredominantly of cetyl and stearyl alcohols); oleyl alcohol;polyoxyethylene glycol alkyl ethers (Brij) such as, octaethylene glycolmonododecyl ether and pentaethylene glycol monododecyl ether;polyoxypropylene glycol alkyl ethers; glucoside alkyl ethers, such asdecyl glucoside, lauryl glucoside and octyl glucoside; polyoxyethyleneglycol octylphenol ethers, such as Triton X-100; polyoxyethylene glycolalkylphenol ethers, such as Nonoxynol-9; glycerol alkyl esters, such asglyceryl laurate; polyoxyethylene glycol sorbitan alkyl esters, such asPolysorbate 20 (Polyoxyethylene (20) sorbitan monolaurate), Polysorbate40 (Polyoxyethylene (20) sorbitan monopalmitate), Polysorbate 60(Polyoxyethylene (20) sorbitan monostearate), and Polysorbate 80(Polyoxyethylene (20) sorbitan monooleate); cocamide ME; cocamide DEA;dodecyldimethylamine oxide; poloxamers; DOC; nonylphenoxypolyethoxylethanol NP-40 (Tergitol-type NP-40); octylphenoxypolyethoxylethanol (Noidet P-40); cetyltrimethylammonium bromide;and any mixtures thereof.

In some embodiments, one ml of the processing buffer can comprise about1 U to about 100 U of a degradative enzyme. In some embodiments, one mlof the processing buffer comprises about 5 U to about 50 U of adegradative enzyme. In one embodiment, one ml of the processing buffercomprises about 10 U of a degradative enzyme. Enzyme unit (U) is an artknown term for the amount of a particular enzyme that catalyzes theconversion of 1 μmol of substrate per minute.

In some embodiments, one ml of the processing buffer can comprise about1 μg to about 10 μg of an anti-coagulant. In some embodiment, one ml ofthe processing buffer can comprise about 1 μg to about 5 μg of ananti-coagulant. In one embodiment, one ml of the processing buffercomprises about 4.6 μg of an anti-coagulant.

In some embodiments, one ml of the processing buffer can comprise about1 mg to about 10 mg of anti-coagulant. In some embodiment, one ml of theprocessing buffer can comprise about 1 mg to about 5 mg ofanti-coagulant. In one embodiment, one ml of the processing buffercomprises about 4.6 mg of anti-coagulant.

Exemplary anti-coagulants include, but are not limited to, heparin,heparin substitutes, salicylic acid, D-phenylalanyl-L-prolyl-L-argininechloromethyl ketone (PPACK), Hirudin, Ancrod (snake venom, Vipronax),tissue plasminogen activator (tPA), urokinase, streptokinase, plasmin,prothrombopenic anticoagulants, platelet phosphodiesterase inhibitors,dextrans, thrombin antagonists/inhibitors, ethylene diamine tetraaceticacid (EDTA), acid citrate dextrose (ACD), sodium citrate, citratephosphate dextrose (CPD), sodium fluoride, sodium oxalate, potassiumoxalate, lithium oxalate, sodium iodoacetate, lithium iodoacetate andmixtures thereof.

Suitable heparinic anticoagulants include heparins or active fragmentsand fractions thereof from natural, synthetic, or biosynthetic sources.Examples of heparin and heparin substitutes include, but are not limitedto, heparin calcium, such as calciparin; heparin low-molecular weight,such as enoxaparin and lovenox; heparin sodium, such as heparin,lipo-hepin, liquaemin sodium, and panheprin; heparin sodiumdihydroergotamine mesylate; lithium heparin; and ammonium heparin.

Suitable prothrombopenic anticoagulants include, but are not limited to,anisindione, dicumarol, warfarin sodium, and the like.

Examples of phosphodiesterase inhibitors suitable for use hereininclude, but are not limited to, anagrelide, dipyridamole,pentoxifyllin, and theophylline.

Suitable dextrans include, but are not limited to, dextran70, such asHYSKON™ (CooperSurgical, Inc., Shelton, Conn., U.S.A.) and MACRODEX™(Pharmalink, Inc., Upplands Vasby, Sweden), and dextran 75, such asGENTRAN™ 75 (Baxter Healthcare Corporation).

Suitable thrombin antagonists include, but are not limited to, hirudin,bivalirudin, lepirudin, desirudin, argatroban, melagatran, ximelagatranand dabigatran.

As used herein, anticoagulants can also include factor Xa inhibitors,factor Ha inhibitors, and mixtures thereof. Various direct factor Xainhibitors are known in the art including, those described in Hirsh andWeitz, Lancet, 93:203-241, (1999); Nagahara et al. Drugs of the Future,20: 564-566, (1995); Pinto et al, 44: 566-578, (2001); Pruitt et al,Biorg. Med. Chem. Lett., 10: 685-689, (2000); Quan et al, J. Med. Chem.42: 2752-2759, (1999); Sato et al, Eur. J. Pharmacol, 347: 231-236,(1998); Wong et al, J. Pharmacol. Exp. Therapy, 292:351-357, (2000).Exemplary factor Xa inhibitors include, but are not limited to,DX-9065a, RPR-120844, BX-807834 and SEL series Xa inhibitors. DX-9065ais a synthetic, non-peptide, propanoic acid derivative, 571 D selectivefactor Xa inhibitor. It directly inhibits factor Xa in a competitivemanner with an inhibition constant in the nanomolar range. See forexample, Herbert et al, J. Pharmacol. Exp. Ther. 276:1030-1038 (1996)and Nagahara et al, Eur. J. Med. Chem. 30(suppl):140s-143s (1995). As anon-peptide, synthetic factor Xa inhibitor, RPR-120844 (Rhone-PoulencRorer), is one of a series of novel inhibitors which incorporate3-(S)-amino-2-pyrrolidinone as a central template. The SEL series ofnovel factor Xa inhibitors (SEL1915, SEL-2219, SEL-2489, SEL-2711:Selectide) are pentapeptides based on L-amino acids produced bycombinatorial chemistry. They are highly selective for factor Xa andpotency in the pM range.

Factor Ha inhibitors include DUP714, hirulog, hirudin, melgatran andcombinations thereof. Melagatran, the active form of pro-drugximelagatran as described in Hirsh and Weitz, Lancet, 93:203-241, (1999)and Fareed et al. Current Opinion in Cardiovascular, pulmonary and renalinvestigational drugs, 1:40-55, (1999).

Generally, salt concentration of the processing buffer can range fromabout 10 mM to about 100 mM. In some embodiments, the processing buffercomprises a salt at a concentration of about 25 mM to about 75 mM. Insome embodiment, the processing buffer comprises a salt at aconcentration of about 45 mM to about 55 mM. In one embodiment, theprocessing buffer comprises a salt at a concentration of about 43 mM toabout 45 mM.

The processing buffer can be made in any suitable buffer solution knownthe skilled artisan. Such buffer solutions include, but are not limitedto, TBS, PBS, BIS-TRIS, BIS-TRIS Propane, HEPES, HEPES Sodium Salt, MES,MES Sodium Salt, MOPS, MOPS Sodium Salt, Sodium Chloride, Ammoniumacetate solution, Ammonium formate solution, Ammonium phosphatemonobasic solution, Ammonium tartrate dibasic solution, BICINE bufferSolution, Bicarbonate buffer solution, Citrate Concentrated Solution,Formic acid solution, Imidazole buffer Solution, MES solution, Magnesiumacetate solution, Magnesium formate solution, Potassium acetatesolution, Potassium acetate solution, Potassium acetate solution,Potassium citrate tribasic solution, Potassium formate solution,Potassium phosphate dibasic solution, Potassium phosphate dibasicsolution, Potassium sodium tartrate solution, Propionic acid solution,STE buffer solution, STET buffer solution, Sodium acetate solution,Sodium formate solution, Sodium phosphate dibasic solution, Sodiumphosphate monobasic solution, Sodium tartrate dibasic solution, TNTbuffer solution, TRIS Glycine buffer solution, TRIS acetate-EDTA buffersolution, Triethylammonium phosphate solution, Trimethylammonium acetatesolution, Trimethylammonium phosphate solution, Tris-EDTA buffersolution, TRIZMA® Base, and TRIZMA® HCL. Alternatively, the processingbuffer can be made in water.

In some embodiments, the processing buffer comprises a mixture ofTriton-X, DNAse I, human plasmin, CaCl₂ and Tween-20. In one embodiment,the processing buffer consists of a mixture of Trirton-X, DNAse I, humanplasmin, CaCl₂ and Tween-20 in a TBS buffer.

In one embodiment, one ml of the processing buffer comprises 100 μl ofTriton-X100, 10 of DNAse (1 U/1 μl), 10 μl of human plasmin at 4.6 mg/mland 870 μl of a mixture of TBS, 0.1% Tween-20 and 50 mM CaCl₂.

Reagents and treatments for processing blood before assaying are alsowell known in the art, e.g., as used for assays on Abbott TDx, AxSYM®,and ARCHITECT® analyzers (Abbott Laboratories), as described in theliterature (see, e.g., Yatscoff et al., Abbott TDx Monoclonal AntibodyAssay Evaluated for Measuring Cyclosporine in Whole Blood, Clin. Chem.36: 1969-1973 (1990), and Wallemacq et al., Evaluation of the New AxSYMCyclosporine Assay: Comparison with TDx Monoclonal Whole Blood and EMITCyclosporine Assays, Clin. Chem. 45: 432-435 (1999)), and/or ascommercially available. Additionally, pretreatment can be done asdescribed in Abbott's U.S. Pat. No. 5,135,875, European Pat. Pub. No. 0471 293, and U.S. Pat. App. Pub. No. 2008/0020401, content of all ofwhich is incorporated herein by reference. It is to be understood thatone or more of these known reagents and/or treatments can be used inaddition to or alternatively to the sample treatment described herein.

In some embodiments, after addition of the processing buffer, the samplecomprises 1% Triton-X, 10 U of DNase, 4.6 mg/ml of plasmin, 5 mMCalcium, 0.01% of Tween 20, 2.5 mM of Tris, 150 mM of NaCl and 0.2 mM ofKCl in addition to the components already present in the sample.

After addition of the processing buffer, the sample can undergo mixing.This can be simply accomplished by agitating the sample, e.g., shakingor vortexing the sample and/or moving the sample around, if it is in amicrofluidic device. In other embodiments where the microbe-targetingsubstrate is in a form of a dipstick or a membrane, themicrobe-targeting dipstick or membrane can be dipped in a volume of atest sample and gently agitated with a rocking motion.

After addition of the processing reagents, the sample can be incubatedfor a period of time, e.g., for at least one minute, at least twominutes, at least three minutes, at least four minutes, at least fiveminutes, at least ten minutes, at least fifteen minutes, at least thirtyminutes, at least forty-five minutes, or at least one hour. Suchincubation can be at any appropriate temperature, e.g., room-temperature(e.g., about 16° C. to about 30° C.), a cold temperature (e.g. about 0°C. to about 16° C.), or an elevated temperature (e.g., about 30° C. toabout 95° C.). In some embodiments, the sample is incubated for aboutfifteen minutes at room temperature.

1206 (1208 (Microbe Capture) and 1210 (Microbe Separation)):

After processing of the sample, the sample can be subjected to a microbecapture process. During the microbe capture process, a microbe-targetingsubstrate added into a test sample can capture one or more microbespresent in the test sample. In some embodiments, the microbe captureprocess can be repeated and/or performed for a sufficient amount of timeto allow for concentrating and/or cleaning up the test sample beforemicrobe detection. Thus, microbe capture and separation processdescribed herein can be used for concentrating and/or cleaning up asample before analysis for a target component in the sample.

In some embodiments, the microbe capture process can comprise mixingnano- and/or micron-sized beads or particles coated with affinitymolecules (e.g., FcMBL or engineered microbe-binding molecules describedherein) which can bind to a microbe in the sample. These affinitymolecule coated nano- and/or micron-sized beads or particles are alsoreferred to as “coated-microbeads” herein. These coated-microbeads canbe magnetic microbeads or non-magnetic microbeads (e.g., fluorescentmicrobeads).

In some embodiments, the coated-microbeads can be microbe-targetingmagnetic microbeads described herein.

Amount of coated-microbeads added to the sample can be dependent on anumber of different factors, such as, number of affinity molecules oneach microbead, size of the microbead, binding affinity of the affinitymolecule to the microbe, and concentration of the microbe in the sample.Additionally, amount of coated-microbeads added to the sample can beadjusted to optimize the capture of microbes. In some embodiments,amount of coated-microbeads added to the sample is such that a microbeadbinds with one microbe. However, each microbe can be bound to more thanone coated-microbeads. This can reduce cross-linking of multiplemicrobes together which can lead to coagulation and/or precipitation ofsuch cross-linked microbes from the sample. Generally, about 100 toabout 10⁹ coated-microbeads can be added to each ml of the sample. Insome embodiments, about 10⁴ to about 5×10⁶ coated-microbeads can beadded for each ml of sample. Stated another way, in some embodiments,the total amount of the microbe-binding molecules contacted with thetest sample can range from about 0.01 μg to about 1 mg, about 0.1 μg toabout 500 μg, about 0.5 μg to about 250 μg, about 1 μg to about 100 μg,or about 3 μg to about 60 μg. In some embodiments, the total amount ofthe microbe-binding molecules contacted with the test sample can rangefrom about 500 μg to about 1000 mg, about 1 mg to about 750 mg, about 5mg to about 500 mg, about 10 mg to about 250 mg, or about 25 mg to about100 mg.

In some embodiments, a plurality of coated-microbeads can be contactedwith a test sample. The plurality of coated-microbeads can comprise atleast two subsets (e.g., 2, 3, 4, 5, or more subsets), wherein eachsubset of coated-microbeads have a pre-determined dimension. In someembodiments, the plurality of coated-microbeads can comprise a firstsubset of the coated-microbeads and a second subset of thecoated-microbeads. In such embodiments, the first subset of thecoated-microbeads each has a first pre-determined dimension; and thesecond subset of the coated-microbeads each has a second pre-determineddimension.

The pre-determined dimension of a coated-microbead depends, in part, onthe dimension of a microbead described herein to which the engineeredmicrobe-binding molecules are conjugated. For example, the microbead canhave a size of about 10 nm to 10 μm, about 20 nm to about 5 μm, about 40nm to about 1 μm, about 50 nm to about 500 nm, or about 50 nm to about200 nm.

Additionally, each subset of the coated-microbeads can comprise on theirsurfaces substantially the same density or different densities of theaffinity molecules (e.g., FcMBL or engineered microbe-binding moleculesdescribed herein).

Different subsets of the plurality of the coated-microbeads can bebrought into contact with a test sample in any manner. For example, insome embodiments, the plurality of the coated-microbeads can be providedas a single mixture comprising at least two subsets of thecoated-microbeads to be added into a test sample. In some embodiments,in order to distinguish among different subsets of thecoated-microbeads, the coated-microbeads in each subset can have adistinct detection label, e.g., a distinctly-fluorescent label that canbe sorted afterward, for example, by flow cytometry.

In other embodiments, the plurality of the coated-microbeads can bebrought into contact with a test sample in a sequential manner. Forexample, a test sample can be contacted with a first subset of thecoated-microbeads, followed by a contact with at least one more subsetsof the coated-microbeads. The previous subset of the coated-microbeadscan be removed from the test sample before addition of another subset ofthe coated-microbeads into the test sample.

In some embodiments, the coated-microbeads are or a microbe-targetingsubstrate is present in the processing buffer. In one embodiment, one mlof the processing buffer comprises 100 μl of Triton-X100, 10 μl of asolution comprising about 25 million microbeads (AKT-FC-MBL on 1 μmMYONE™ C1 streptavidin microbeads), 10 μl of DNAse (1 U/1 μl), 10 μl ofhuman plasmin at 4.6 mg/ml and 870 μl of a mixture of TBS, 0.1%Tween-20. In some embodiments, the processing buffer can include acalcium salt, e.g., CaCl₂ (e.g., ˜50 mM CaCl₂). In some embodiments, theprocessing or capture buffer can include no calcium salt, e.g., CaCl₂.

After addition of the coated-microbeads, the coated-microbeads can bemixed in the sample to allow microbes to bind with the microbeads. Thiscan be simply accomplished by agitating the sample, e.g., shaking orvortexing the sample and/or moving the sample around in a microfluidicdevice. In other embodiments where the microbe-targeting substrate is ina form of a dipstick or a membrane, the microbe-targeting dipstick ormembrane can be dipped in a volume of a test sample and gently agitatedwith a rocking motion.

The volume of a test sample required for contacting themicrobe-targeting substrate can vary with, e.g., the selection of themicrobe-targeting substrate (e.g., microbeads, fibers, filters, filters,fibers, screens, mesh, tubes, hollow fibers), the concentration ofmicrobes present in a test sample, and/or the platform used to carry outthe assay (e.g., a microfluidic device or a blood collection tube, amicrotiter plate). In some embodiments, the test sample volume used toperform the assay described herein, e.g., in a microfluidic platform,can range from about 1 μL to about 500 μL, from about 5 μL to about 250μL, or from about 10 μL to about 100 μL. In other embodiments, the testsample volume used to perform the assay described herein, e.g., in atube platform, can range from about 0.05 mL to about 50 mL, from about0.25 ml to about 50 ml, about 0.5 ml to about 25 ml, about 1 ml to about15 ml, or about 2 ml to about 10 ml. In some embodiments, the testsample volume used to perform the assay described herein can be about 1mL to about 5 ml. In one embodiment, the test sample volume used toperform the assay described herein is about 5 ml to about 10 mL.

After addition of the microbe-targeting substrate (e.g.,coated-microbeads) into a test sample (containing a processing buffer),the sample mixture can be incubated for a period of time to allow themicrobe of interest to bind onto the microbe-targeting substrate, e.g.,incubation for at least one minute, at least two minutes, at least threeminutes, at least four minutes, at least five minutes, at least tenminutes, at least fifteen minutes, at least about twenty minutes, atleast thirty minutes, at least forty-five minutes, or at least one hour.In one embodiment, the sample mixture can be incubated for a period ofabout 10-20 minutes. Such incubation can be performed at any appropriatetemperature, e.g., room-temperature (e.g., about 16° C. to about 30°C.), a cold temperature (e.g. about 0° C. to about 16° C.), or anelevated temperature (e.g., about 30° C. to about 95° C.). In someembodiments, the incubation can be performed at a temperature rangingfrom about room temperature to about 37° C. In some embodiments, thesample can be incubated for about 10 mins to about 20 mins at roomtemperature. In some embodiments, the sample is incubated for aboutfifteen minutes at room temperature.

To prevent or reduce agglutination (or non-specific binding) duringseparation of the microbes from the sample, additional reagents can beadded to the sample mixture. Such reagents are also referred to asblocking reagents herein. For example, these blocking reagents cancomprise a ligand of the affinity molecules on the coated-microbeads.Addition of such blocking reagents can reduce agglutination by bindingwith any empty ligand binding sites on the affinity molecules.Accordingly, when microbe-targeting magnetic microbeads are used forcapturing the microbes, the blocking reagent can be a carbohydrate, suchas mannose. Amount of additional reagent can depend on the amount ofmicrobeads added to the sample. Generally, about the reagent is added toa final concentration of about 0.1 mM to about 10 mM. The amount of theblocking agent required can vary, at least partly, with the amountand/or surface area of the microbe-targeting substrate that is incontact with a test sample. In some embodiments, the blocking reagentcan be added to a final concentration of about 0.1% (w/v) to about 10%(w/v), about 0.5% (w/v) to about 7.5% (w/v), or about 1% (w/v) to about5% (w/v). In some embodiments, about 1% casein can be used as a blockingagent in the assay described herein.

After addition of the blocking reagent, the sample mixture can beincubated for a period of time to allow the blocking reagent to bind towith the affinity molecules, e.g., for at least one minute, at least twominutes, at least three minutes, at least four minutes, at least fiveminutes, at least ten minutes, at least fifteen minutes, at least thirtyminutes, at least forty-five minutes, or at least one hour. Suchincubation can be at any appropriate temperature, e.g., room-temperature(e.g., about 16° C. to about 30° C.), a cold temperature (e.g. about 0°C. to about 16° C.), or an elevated temperature (e.g., about 30° C. toabout 95° C.). In some embodiments, the sample is incubated for aboutfifteen minutes at room temperature. In some embodiments, incubation isfor about 5 seconds to about 60 seconds. In some embodiments, theincubation can be performed at a temperature ranging from about roomtemperature to about 37° C. In some embodiments, the sample is incubatedfor about fifteen minutes at room temperature.

To prevent or reduce non-specific binding during the contact between amicrobe-targeting substrate and a test sample, in some embodiments, themicrobe-targeting substrate (e.g., coated-microbeads) and/or the testsample can be pre-treated with a blocking agent that does not react withmicrobes, e.g., casein, normal serum, BSA, non-fat dry milk powder andany art-recognized block agent, before contacting each other.Optionally, microbe-targeting substrate after blocking can be washedwith any art-recognized buffer to remove any leftover blocking agent.The number of wash steps can range from 1 to many, e.g., 1, 2, 3, 4, 5,6, 7, 8, 9, 10 or more wash steps. In one embodiment, themicrobe-targeting substrate after blocking can be washed with a buffer,e.g., TBST, for about at least 1-3 times.

Exemplary Optional Modifications to 1208 (Microbe Capture):

In accordance with one aspect described herein, the test sample can becontacted with a microbe-targeting substrate in the presence of achelating agent. Without wishing to be bound by theory, the addition ofa chelating agent to a test sample and/or processing buffer can reducethe likelihood of any protein A- and protein G-negative microbe (e.g.,E. coli), but not protein A- or protein G-expressing microbe (e.g., S.aureus) in the test sample, to bind with at least one microbe-bindingmolecule. Accordingly, detection of any microbes bound on themicrobe-targeting substrate described herein in the presence of achelating agent can determine the presence or absence of a protein A- orprotein G-expressing microbe in a test sample.

The chelating agent can be added into the processing buffer comprisingthe test sample. The amount of the chelating agent is sufficient tochelate free calcium ions and thus prevent or reduce calcium-dependentcarbohydrate recognition domain binding (e.g., mannose-binding lectin)with a microbe. The amount of the chelating agent needed to prevent orreduce calcium-dependent carbohydrate recognition domain binding (e.g.,mannose-binding lectin) with a microbe can depend on, e.g., theconcentration of free calcium ions present in a test sample andoptionally a capture buffer, e.g., used to dilute a chelating agentand/or a test sample. Thus, in some embodiments, the concentration ofthe chelating agent can be higher than the total concentration of freecalcium ions present in the combined solution of a test sample and acapture buffer. For example, in some embodiments, the concentration ofthe chelating agent can be at least about 30% higher, including at leastabout 40%, at least about 50%, at least about 60%, at least about 70%,at least about 80%, at least about 90%, at least about 95%, at leastabout 98%, up to and including 100%, or any percent between about 30%and about 100%, higher than the total concentration of free calcium ionspresent in the combined solution of a test sample and a capture buffer.In other embodiments, the concentration of the chelating agent can be atleast about 1.5-fold, at least about 2-fold, at least about 3-fold, atleast about 4-fold, at least about 5-fold, at least about 6-fold, atleast about 7-fold, at least about 8-fold, at least about 9-fold, atleast about 10-fold, at least about 15-fold, at least about 20-fold, atleast about 30-fold, at least about 40-fold, at least about 50-fold, atleast about 75-fold, at least about 100-fold or more, higher than thetotal concentration of free calcium ions present in the combinedsolution of a test sample and a capture buffer. In one embodiment, theconcentration of the chelating agent can be at least about 5-fold toabout 50-fold, or at least about 7-fold to about 25-fold, higher thanthe total concentration of free calcium ions present in the combinedsolution of a test sample and a capture buffer.

In some embodiments, the concentration of a chelating agent present inthe test sample and optionally a processing or capture buffer, e.g.,used to dilute the chelating agent and/or the test sample, can rangefrom about 0.1 mM to about 1 M, about 10 mM to about 500 mM, about 20 mMto about 250 mM, or about 25 mM to about 125 mM. In one embodiment, theconcentration of a chelating agent present in the test sample andoptionally a capture buffer can be about 25 mM to about 125 mM.

In some embodiments, the concentration of a chelating agent present inthe test sample containing the microbe-targeting substrate can besufficient to reduce the likelihood of a protein A- and proteinG-negative microbe (e.g., E. coli), if present in the test sample, tobind with at least one microbe-binding molecule. For example, theconcentration of a chelating agent present in the test sample with themicrobe-targeting substrate can be sufficient to reduce the number ofprotein A- and protein G-negative microbes (e.g., E. coli), if presentin the test sample, to bind with at least one microbe-binding molecule,by at least about 30%, at least about 40%, at least about 50%, at leastabout 60%, at least about 70%, at least 80% or higher, as compared tothe number of protein A- and protein G-negative microbes (e.g., E. coli)bound on the microbe binding molecules in the absence of the chelatingagent. In some embodiments, the concentration of a chelating agentpresent in the test sample with the microbe-targeting substrate can besufficient to reduce the number of protein A- and protein G-negativemicrobes (e.g., E. coli), if present in the test sample, to bind with atleast one microbe-binding molecule, by at least about 85%, at leastabout 90%, at least about 95%, at least about 98%, at least about 99%,up to and including 100%, or any values between about 85% and about100%, as compared to the number of protein A- and protein G-negativemicrobes (e.g., E. coli) bound on the microbe-binding molecules in theabsence of the chelating agent.

The protein A-expressing and protein G-expressing microbes can generallybind to microbe-binding molecules via two independent (but additive)mechanisms: Fc-mediated binding and microbe surface-binding domain(e.g., MBL)-mediated binding. Without wishing to be bound by theory,while the protein A-expressing and protein G-expressing microbes canstill be captured on the microbe-targeting substrate in the presence ofa chelating agent, the presence of free calcium ions can furtherincrease the number of protein A-expressing and protein G-expressingmicrobes bound to the microbe-targeting substrate, because the overallbinding in the presence of calcium ions can be almost twice as strong asin the absence of calcium ions.

Accordingly, in some embodiments, the concentration of a chelating agentpresent in the test sample containing the microbe-targeting substratecan reduce the number of protein A-expressing microbes and/or proteinG-expressing microbes bound onto the microbe-targeting substrate, butsuch effect as compared to that on the protein A- and protein G-negativemicrobes (e.g., E. coli) is much smaller, e.g., at least about 30%smaller, at least about 40% smaller, at least about 50%, at least about60% smaller, at least about 70% smaller, or at least about 80% smaller.For example, as shown in FIG. 29, while the concentration of a chelatingagent (e.g., 100 mM EDTA) is sufficient to reduce the binding of proteinA- and protein G-negative microbes (e.g., E. coli) with amicrobe-targeting substrate (e.g., a microbe-targeting membrane) to anundetectable level, there is still a detectable level of proteinA-expressing microbes (e.g., S. aureus) binding to the microbe-targetingmembrane. Therefore, in some embodiments, the concentration of achelating agent used in the assay described herein should be high enoughto prevent at least about 80% or higher, including at least about 90%,at least about 95%, up to and including 100%, of the protein A- andprotein G-negative microbes (e.g., E. coli) from binding to bemicrobe-targeting substrate, but low enough to allow at least about 30%or higher, including at least about 40%, at least about 50%, at leastabout 60%, at least about 70% or higher, of the protein A-expressingmicrobes (e.g., S. aureus) or protein G-expressing microbes to bind withthe microbe-targeting substrate. In one embodiment, the concentration ofa chelating agent used in the assay described herein should be highenough to prevent at least about 90% or higher, of the protein A- andprotein G-negative microbes (e.g., E. coli), if any present in the testsample, from binding to be microbe-targeting substrate, but low enoughto allow at least about 50% of the protein A-expressing microbes (e.g.,S. aureus) or protein G-expressing microbes, if any present in the testsample, to bind with the microbe-targeting substrate.

Examples of calcium ion-chelating agents can include, but are notlimited to, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid,ethylenediaminetetraacetic acid (EDTA); ethyleneglycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid; ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA),1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), abuffer containing citrate,N,N-Bis(2-(bis-(carboxymethyl)amino)ethyl)-glycine (DTPA),nitrilo-2,2′,2″-triacetic acid (NTA), a buffer that precipitates acalcium ion from the test sample, including, e.g., a phosphate buffer, acarbonate buffer and a bicarbonate buffer, a low pH buffer (e.g., a pHbuffer less than pH 7 or less than pH 6), citric acids and its salts,gluconic acid and its salts, alkali metal pyrophosphates, alkali metalpolyphosphates, sodium hexametaphosphate, triethylene tetramine,diethylene triamine, o-phenanthroline, oxalic acid and any combinationsthereof.

The chelating agent can be directly added to the test sample or preparedin a processing or capture buffer, which is then added to the testsample in contact with the microbe-targeting substrate. The processingor capture buffer can be any buffered solutions, e.g., with a pH rangingfrom about 6 to about 10. In some embodiments, the processing or capturebuffer can include, but is not limited to, a tris-buffered saline, aphosphate buffered saline or a combination thereof. In some embodiments,the processing or capture buffer can include a surfactant, e.g., toprevent non-specific binding of a microbe to a microbe-surface-bindingdomain of the microbe-targeting substrate, and/or to saturatenon-specific binding sites, if any, present in the microbe-targetingsubstrate. A surfactant or detergent, e.g., as described earlier, can bedissolved in a buffered solution in any amount, e.g., ranging from about0.001% (v/v) to about 5% (v/v), from about 0.01% (v/v) to about 2.5%(v/v), or from about 0.05% (v/v) to about 1% (v/v). In some embodiments,the surfactant added to the processing or capture buffer can includeTween 80 or polysorbate 80 at a concentration of about 0.01% to about0.1%. In one embodiment, the surfactant added to the processing orcapture buffer can include Tween 80 or polysorbates 80 at aconcentration of about 0.05%.

After incubation, the microbe-targeting substrate can then be analyzed,as described below, for the presence or absence of a bound microbe. Inthe absence of a microbe-targeting substrate-bound microbe, in someembodiments, the previous volume of the test sample or a new freshvolume of the test sample can be contacted with a freshmicrobe-targeting substrate in the presence of free calcium ions, e.g.,to determine the presence or absence of protein A- and proteinG-negative microbes (e.g., E. coli). In some embodiments, the freecalcium ions can be produced adding a sufficient amount of calcium saltsin the test sample. If there has been a chelating agent present in thetest sample, a higher amount of calcium salts is generally needed inorder to obtain free calcium ions.

As used herein, the term “free calcium ions” refers to calcium ions thatare not complexed with any molecule or compound, e.g., a chelatingagent, which can hinder its reaction with other molecules or ions tomediate binding of carbohydrate patterns on a microbial cell surface toa microbe surface-binding domain (e.g., MBL) of the engineeredmicrobe-binding molecule. Accordingly, in some embodiments, free calciumions can be present in the absence of chelating agent. In someembodiments, free calcium ions can be present in a solution comprising achelating agent and calcium ions, wherein the amount of calcium ionspresent in the solution is at least about 30% more than an amountsufficient to interact with substantially all the chelating agentmolecules present in the solution to form chelate complexes. Forexample, in some embodiments, in order to obtain free calcium ions, theamount of calcium ions present in the solution can be at least about30%, including at least about 40%, at least about 50%, at least about60%, at least about 70%, at least about 80%, at least about 90%, atleast about 95%, at least about 98%, up to and including 100% and anypercent between 30% and 100%, more than an amount sufficient to interactwith substantially all the chelating agent molecules present in thesolution to form chelate complexes. In some embodiments, in order toobtain free calcium ions, the amount of calcium ions present in thesolution can be at least about 1-fold, at least about 2-fold, at leastabout 3-fold, at least about 4-fold, at least about 5-fold, at leastabout 6-fold, at least about 7-fold, at least about 8-fold, at leastabout 9-fold, at least about 10-fold, at least about 15-fold, at leastabout 20-fold, at least about 25-fold, at least about 50-fold, at leastabout 100-fold, at least about 500-fold, at least about 1000-fold, morethan an amount sufficient to interact with substantially all thechelating agent molecules present in the solution to form chelatecomplexes. In some embodiments, free calcium ions can be present in asolution when the concentration of calcium ions in the solution is atleast about 1.5-fold, at least about 2-fold, at least about 3-fold, atleast about 4-fold, at least about 5-fold, at least about 6-fold, atleast about 7-fold, at least about 8-fold, at least about 9-fold, atleast about 10-fold, at least about 20-fold, or higher than theconcentration of a chelating agent present in the same solution.

In some embodiments, calcium ions can be obtained from a water-solublecalcium salt. By the term “water-soluble calcium salt” is meant acalcium salt which has significant solubility in water at roomtemperature, for example at least 1 gram per 100 ml water, at least 10grams per 100 ml water, or at least 25 grams per 100 ml water or higher.Examples of calcium salts include, without limitations, calciumchloride, calcium fluoride, calcium bromide, calcium iodide, calciumnitrate, calcium citrate, calcium formate, calcium acetate, calciumgluconate, calcium ascorbate, calcium lactate, calcium glycinate andmixtures thereof. In some embodiments, calcium chloride can be used as asource of calcium ions.

Free calcium ions can be present at a concentration or an amountsufficient to mediate binding of calcium-dependent carbohydraterecognition domain with a microbe surface. In some embodiments, freecalcium ions can be present at a concentration of at least about 1 μM,at least about 10 μM, at least about 25 μM, at least about 50 μM, atleast about 100 μM, at least about 250 μM, at least about 500 μM, or atleast about 1 mM or higher. In some embodiments, the free calcium ionscan be present at a concentration of at least about 1 mM, at least about2.5 mM, at least about 5 mM, at least about 10 mM, at least about 25 mM,at least about 50 mM, at least about 75 mM, at least about 100 mM orhigher. In other embodiments, the free calcium ions can be present at aconcentration of at least about 100 mM, at least about 150 mM, at leastabout 200 mM, at least about 300 mM, at least about 400 mM, at leastabout 500 mM, at least about 600 mM, at least about 700 mM, at leastabout 800 mM, at least about 900 mM, at least about 1 M or higher. Inone embodiment, the free calcium ions can be present at a concentrationof about 1 mM to about 10 mM. In one embodiment, the free calcium ionscan be present at a concentration of at least about 5 mM.

While a chelating agent can be added during an initial capture of amicrobe on a microbe-targeting substrate, the chelating agent can alsobe first excluded to allow the initial capture of any microbe, includingprotein A- and protein G-negative microbes, on a microbe-targetingsubstrate in the presence of free calcium ions, but added after thecapture to remove any captured protein A- or protein G-negative microbesfrom the microbe-targeting substrate.

Accordingly, in some embodiments, the microbe capture can comprise (i)contacting at least a first volume of a test sample with amicrobe-targeting substrate described herein in the presence of freecalcium ions, and (ii) contacting the microbe-binding molecule of themicrobe-targeting substrate described herein, upon the contact with thetest sample, with a solution comprising a chelating agent.

When the microbe-targeting substrate is contacted with a test sample inthe presence of free calcium ions as described herein, microbes thatprimarily depend on calcium-dependent MBL-mediated binding such asprotein A- and protein G-negative microbes, e.g., E. coli can bind tothe microbe-target substrate, in addition to microbes associated withFc-mediated binding such as protein A-expressing microbes (e.g., S.aureus), and protein G-expressing microbes.

To elute off or remove from the microbe-targeting substrate the capturedmicrobes that primarily depend on calcium-dependent MBL-mediated bindingsuch as protein A- and protein G-negative microbes, e.g., E. coli, themicrobe-binding molecules on the microbe-targeting substrates can becontacted with a solution comprising a sufficient amount of a chelatingagent as described herein. The solution comprising the chelating agentcan be same as a capture buffer described above. In such embodiments,the microbe-targeting substrate can be incubated with the solutioncomprising a chelating agent for a period of time to allow microbes thatprimarily bind to microbe-binding molecules via calcium-dependentMBL-mediated binding to elute off the microbe-targeting substrate, e.g.,incubation for at least one minute, at least two minutes, at least threeminutes, at least four minutes, at least five minutes, at least tenminutes, at least fifteen minutes, at least thirty minutes, at leastforty-five minutes, or at least one hour. Such incubation can beperformed at any appropriate temperature, e.g., room-temperature (e.g.,about 16° C. to about 30° C.), a cold temperature (e.g. about 0° C. toabout 16° C.), or an elevated temperature (e.g., about 30° C. to about95° C.). In some embodiments, the microbe-targeting substrate can beincubated with the solution comprising a chelating agent for at leastabout 5 mins to about 15 mins at room temperature.

In these embodiments, the concentration of a chelating agent used in theassay described herein is sufficient to elute off or remove from themicrobe-targeting substrate at least about 30% of the bound protein A-and protein G-negative microbes (e.g., E. coli). For example, theconcentration of a chelating agent used in the assay described herein issufficient to elute off or remove from the microbe-targeting substrateat least about 30% of the bound protein A- and protein G-negativemicrobes (e.g., E. coli), including at least about 40%, at least about50%, at least about 60%, at least about 70%, at least 80% or higher, ofthe bound protein A- and protein G-negative microbes (e.g., E. coli). Insome embodiments, the concentration of a chelating agent used in theassay described herein is sufficient to elute off or remove from themicrobe-targeting substrate at least about 85% of the bound protein A-and protein G-negative microbes (e.g., E. coli), including at leastabout 85%, at least about 90%, at least about 95%, at least about 98%,up to and including 100%, or any values between about 85% and about100%, of the bound protein A- and protein G-negative microbes (e.g., E.coli).

As noted above, the protein A-expressing and protein G-expressingmicrobes can bind to microbe-binding molecules via Fc-mediated andcalcium ion-dependent MBL-mediated binding. Without wishing to be boundby theory, the concentration of a chelating agent used in the assaydescribed herein can also elute off or remove at least a portion of theprotein A-expressing and/or protein G-expressing microbes from themicrobe-targeting substrate. For example, the concentration of achelating agent used to elute off or remove protein A- and proteinG-negative microbes from the microbe-targeting substrate can besufficient to elute off or remove no more than 60%, no more than 50%, nomore than 40%, no more than 30%, no more than 20%, no more than 10% orlower, of the bound protein A-expressing and/or protein G-expressingmicrobes. In some embodiments, the concentration of a chelating agentused to elute off or remove from the microbe-targeting substrate atleast about 80% or more, including at least about 90% or more, of thebound protein A- and protein G-negative microbes can be sufficient toelute off or remove no more than 50%, or more than 40% of the boundprotein A-expressing and/or protein G-expressing microbes. As shown inFIG. 29, while the concentration of a chelating agent (e.g., 100 mMEDTA) is sufficient to elute off or remove substantially all protein A-and protein G-negative microbes (e.g., E. coli) from a microbe-targetingsubstrate to an undetectable level, there is still a detectable level ofprotein A-expressing microbes (e.g., S. aureus) remained bound to themicrobe-targeting membrane.

As a person having ordinary skill in the art can appreciate, the assaydescribed herein can further comprise isolating the microbe-targetingsubstrate from the test sample, e.g., as described below, beforecontacting microbe-binding molecules on its substrate surface with thesolution comprising the chelating agent described herein.

1210 (Microbe Separation from Sample):

The sample mixture is then subjected to a microbe separation process. Insome embodiments, because microbes are bound with one or more magneticmicrobeads, a magnet can be employed to separate the bound microbes fromthe test sample. The skilled artisan is well aware of methods forcarrying out magnetic separations. Generally, a magnetic field gradientcan be applied to direct the capture of magnetic microbeads. Optionally,the bound microbe can be washed with a buffer to remove any leftoversample and unbound components. Number of wash steps can range from 1 tomany, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more wash steps. Withoutwishing to be bound by a theory, capture and separation of the boundmicrobes from the sample can concentrate the microbes and also removecomponents, which can interfere with the assay or process, from the testsample.

The magnetic field source can be any magnet device positioned togenerate the magnetic field gradient that is used to pull the capturedmicrobe out from the sample. An electromagnetic controller can be usedto control and adjust the magnetic field and gradients thereof, and tocontrol the migration, separation and orientation of the magneticallybound microbes. The magnetic field gradient can be generated by apermanent magnet or by an electromagnetic signal generator. Theelectromagnetic signal generator can include an electromagnet orelectrically-polarizable element, or at least one permanent magnet. Themagnetic field gradient can be produced at least in part according to apre-programmed pattern. The magnetic field gradient can have a definedmagnetic field strength and/or spatial orientation. In some embodiments,the magnetic field gradient has a defined magnetic field strength. Theterm “magnetic field gradient” as used herein refers to a variation inthe magnetic field with respect to position. By way of example only, aone-dimensional magnetic field gradient is a variation in the magneticfield with respect to one direction, while a two-dimensional magneticfield gradient is a variation in the magnetic field with respect to twodirections.

As used herein, the term “magnetic field” refers to magnetic influenceswhich create a local magnetic flux that flows through a composition andcan refer to field amplitude, squared-amplitude, or time-averagedsquared-amplitude. It is to be understood that magnetic field can be adirect-current (DC) magnetic field or alternating-current (AC) magneticfield. The magnetic field strength can range from about 0.00001 Teslaper meter (T/m) to about 10⁵ T/m. In some embodiments, the magneticfield strength can range from about 0.0001 T/m to about 10⁴ T/m. In someother embodiments, the magnetic field strength can range from about0.001 T/m to about 10³ T/m.

In some embodiments, microbe capture and/or microbe-targeting substrateseparation can be performed by a rapid microbe diagnostic device asdescribed in Int. Pat. App. No. WO 2011/091037, filed Jan. 19, 2011, thecontent of which is incorporated herein by reference. A rapid microbediagnostic device as described in Int. Pat. App. No. WO 2011/091037,filed Jan. 19, 2011, can be modified to replace the capture chamber orcapture and visualization chamber with an s-shaped flow path. A magnetcan then be used to capture bound microbe against the flow path wall;separating the bound microbe from rest of the sample.

In some embodiments, microbe capture and/or separation is by a device ormethod as described in U.S. Pat. App. Pub. No. 2009/0220932, No.2009/007861, No. 2010/0044232, No. 2007/0184463, No. 2004/0018611, No.2008/0056949, No. 2008/0014576, No. 2007/0031819, No. 2008/0108120, andNo. 2010/0323342, the contents of which are all incorporated herein byreference.

Without limitations, if a microbe-targeting substrate does not possess amagnetic property, isolation of a microbe-targeting substrate (e.g.,particles, posts, fibers, dipsticks, membrane, filters, capillary tubes,etc.) from the test sample can be carried out by non-magnetic means,e.g., centrifugation, and filtration. In some embodiments where themicrobe-targeting substrate is in a form a dipstick or membrane, themicrobe-targeting dipstick or membrane can be simply removed from thetest sample, where microbes, if any, in the test sample, remained boundto the engineered microbe-binding molecules conjugated to the dipstickor membrane substrate.

Optionally, the microbe-targeting substrate after isolated from the testsample or processing buffer can be washed with a buffer (e.g., TBST) toremove any residues of test sample, solution comprising the chelatingagent or any unbound microbes. The number of wash steps can range from 1to many, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more wash steps. In oneembodiments, the microbe-targeting substrate after isolated from thesolution comprising the chelating agent and/or the test sample can bewashed with a buffer (e.g., TBST) for about at least 1-3 times.

1212 (Microbe Detection/Analysis):

A detection component, device or system can be used to detect and/oranalyze the presence of the separated microbe, for example, byspectroscopy, electrochemical detection, polynucleotide detection,fluorescence anisotropy, fluorescence resonance energy transfer,electron transfer, enzyme assay, magnetism, electrical conductivity,isoelectric focusing, chromatography, immunoprecipitation,immunoseparation, aptamer binding, filtration, electrophoresis, use of aCCD camera, immunoassay, ELISA, Gram staining, immunostaining,microscopy, immunofluorescence, western blot, polymerase chain reaction(PCR), RT-PCR, fluorescence in situ hybridization, sequencing, massspectroscopy, or substantially any combination thereof. The separatedmicrobe can remain bound on the microbe-targeting substrate duringdetection and/or analysis, or be isolated form the microbe-targetingsubstrate prior to detection and/or analysis.

In some embodiments, labeling molecules that can bind with the microbecan also be used to label the microbes for detection. As used herein, a“labeling molecule” refers to a molecule that comprises a detectablelabel and can bind with a target microbe. Labeling molecules caninclude, but are not limited to, MBL or a portion thereof, FcMBL,AKT-FcMBL, wheat germ agglutinin, lectins, antibodies (e.g.,gram-negative antibodies or gram-positive antibodies, antibiotics tospecific microbial strains or species), antigen binding fragments ofantibodies, aptamers, ligands (agonists or antagonists) of cell-surfacereceptors and the like. The labeling molecule can also be a non-specificlabeling molecule that non-specifically stains all viable cells in asample.

As used herein, the term “detectable label” refers to a compositioncapable of producing a detectable signal indicative of the presence of atarget. Detectable labels include any composition detectable byspectroscopic, photochemical, biochemical, immunochemical, electrical,optical or chemical means. Suitable labels include fluorescentmolecules, radioisotopes, nucleotide chromophores, enzymes, substrates,chemiluminescent moieties, bioluminescent moieties, and the like. Assuch, a label is any composition detectable by spectroscopic,photochemical, biochemical, immunochemical, electrical, optical orchemical means needed for the methods and devices described herein.

A wide variety of fluorescent reporter dyes are known in the art.Typically, the fluorophore is an aromatic or heteroaromatic compound andcan be a pyrene, anthracene, naphthalene, acridine, stilbene, indole,benzindole, oxazole, thiazole, benzothiazole, cyanine, carbocyanine,salicylate, anthranilate, coumarin, fluorescein, rhodamine or other likecompound.

Exemplary fluorophores include, but are not limited to, 1,5 IAEDANS;1,8-ANS; 4-Methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein;5-Carboxyfluorescein (5-FAM); 5-Carboxynapthofluorescein (pH 10);5-Carboxytetramethylrhodamine (5-TAMRA); 5-FAM (5-Carboxyfluorescein);5-Hydroxy Tryptamine (HAT); 5-ROX (carboxy-X-rhodamine); 5-TAMRA(5-Carboxytetramethylrhodamine); 6-Carboxyrhodamine 6G; 6-CR 6G; 6-JOE;7-Amino-4-methylcoumarin; 7-Aminoactinomycin D (7-AAD);7-Hydroxy-4-methylcoumarin; 9-Amino-6-chloro-2-methoxyacridine; ABQ;Acid Fuchsin; ACMA (9-Amino-6-chloro-2-methoxyacridine); AcridineOrange; Acridine Red; Acridine Yellow; Acriflavin; Acriflavin FeulgenSITSA; Aequorin (Photoprotein); Alexa Fluor 350™; Alexa Fluor 430™;Alexa Fluor 488™; Alexa Fluor 532™; Alexa Fluor 546™; Alexa Fluor 568™;Alexa Fluor 594™; Alexa Fluor 633™; Alexa Fluor 647™; Alexa Fluor 660™;Alexa Fluor 680™; Alizarin Complexon; Alizarin Red; Allophycocyanin(APC); AMC, AMCA-S; AMCA (Aminomethylcoumarin); AMCA-X; AminoactinomycinD; Aminocoumarin; Anilin Blue; Anthrocyl stearate; APC-Cy7; APTS;Astrazon Brilliant Red 4G; Astrazon Orange R; Astrazon Red 6B; AstrazonYellow 7 GLL; Atabrine; ATTO-TAG™ CBQCA; ATTO-TAG™ FQ; Auramine;Aurophosphine G; Aurophosphine; BAO 9 (Bisaminophenyloxadiazole); BCECF(high pH); BCECF (low pH); Berberine Sulphate; Beta Lactamase; BFP blueshifted GFP (Y66H); BG-647; Bimane; Bisbenzamide; Blancophor FFG;Blancophor SV; BOBO™-1; BOBO™-3; Bodipy 492/515; Bodipy 493/503; Bodipy500/510; Bodipy 505/515; Bodipy 530/550; Bodipy 542/563; Bodipy 558/568;Bodipy 564/570; Bodipy 576/589; Bodipy 581/591; Bodipy 630/650-X; Bodipy650/665-X; Bodipy 665/676; Bodipy Fl; Bodipy FL ATP; Bodipy Fl-Ceramide;Bodipy R6G SE; Bodipy TMR; Bodipy TMR-X conjugate; Bodipy TMR-X, SE;Bodipy TR; Bodipy TR ATP; Bodipy TR-X SE; BO-PRO™-1; BO-PRO™-3;Brilliant Sulphoflavin FF; Calcein; Calcein Blue; Calcium Crimson™;Calcium Green; Calcium Green-1 Ca²⁺ Dye; Calcium Green-2 Ca²⁺; CalciumGreen-5N Ca²⁺; Calcium Green-C18 Ca²⁺; Calcium Orange; Calcofluor White;Carboxy-X-rhodamine (5-ROX); Cascade Blue™; Cascade Yellow;Catecholamine; CFDA; CFP-Cyan Fluorescent Protein; Chlorophyll;Chromomycin A; Chromomycin A; CMFDA; Coelenterazine; Coelenterazine cp;Coelenterazine f; Coelenterazine fcp; Coelenterazine h; Coelenterazinehcp; Coelenterazine ip; Coelenterazine O; Coumarin Phalloidin; CPMMethylcoumarin; CTC; Cy2™; Cy3.1 8; Cy3.5™; Cy3™; Cy5.1 8; Cy5.5™; Cy5™; Cy7™; Cyan GFP; cyclic AMP Fluorosensor (FiCRhR); d2; Dabcyl; Dansyl;Dansyl Amine; Dansyl Cadaverine; Dansyl Chloride; Dansyl DHPE; Dansylfluoride; DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3; DCFDA; DCFH(Dichlorodihydrofluorescein Diacetate); DDAO; DHR (Dihydorhodamine 123);Di-4-ANEPPS; Di-8-ANEPPS (non-ratio); DiA (4-Di-16-ASP); DIDS;Dihydorhodamine 123 (DHR); DiO (DiOC18(3)); DiR; DiR (DiIC18(7));Dopamine; DsRed; DTAF; DY-630-NHS; DY-635-NHS; EBFP; ECFP; EGFP; ELF 97;Eosin; Erythrosin; Erythrosin ITC; Ethidium homodimer-1 (EthD-1);Euchrysin; Europium (III) chloride; Europium; EYFP; Fast Blue; FDA;Feulgen (Pararosaniline); FITC; FL-645; Flazo Orange; Fluo-3; Fluo-4;Fluorescein Diacetate; Fluoro-Emerald; Fluoro-Gold(Hydroxystilbamidine); Fluor-Ruby; FluorX; FM 1-43™; FM 4-46; Fura Red™(high pH); Fura-2, high calcium; Fura-2, low calcium; Genacryl BrilliantRed B; Genacryl Brilliant Yellow 10GF; Genacryl Pink 3G; Genacryl Yellow5GF; GFP (S65T); GFP red shifted (rsGFP); GFP wild type, non-UVexcitation (wtGFP); GFP wild type, UV excitation (wtGFP); GFPuv;Gloxalic Acid; Granular Blue; Haematoporphyrin; Hoechst 33258; Hoechst33342; Hoechst 34580; HPTS; Hydroxycoumarin; Hydroxystilbamidine(FluoroGold); Hydroxytryptamine; Indodicarbocyanine (DiD);Indotricarbocyanine (DiR); Intrawhite Cf; JC-1; JO-JO-1; JO-PRO-1;LaserPro; Laurodan; LDS 751; Leucophor PAF; Leucophor SF; Leucophor WS;Lissamine Rhodamine; Lissamine Rhodamine B; LOLO-1; LO-PRO-1; LuciferYellow; Mag Green; Magdala Red (Phloxin B); Magnesium Green; MagnesiumOrange; Malachite Green; Marina Blue; Maxilon Brilliant Flavin 10 GFF;Maxilon Brilliant Flavin 8 GFF; Merocyanin; Methoxycoumarin; MitotrackerGreen FM; Mitotracker Orange; Mitotracker Red; Mitramycin;Monobromobimane; Monobromobimane (mBBr-GSH); Monochlorobimane; MPS(Methyl Green Pyronine Stilbene); NBD; NBD Amine; Nile Red;Nitrobenzoxadidole; Noradrenaline; Nuclear Fast Red; Nuclear Yellow;Nylosan Brilliant Iavin E8G; Oregon Green™; Oregon Green 488-X; OregonGreen™ 488; Oregon Green™ 500; Oregon Green™ 514; Pacific Blue;Pararosaniline (Feulgen); PE-Cy5; PE-Cy7; PerCP; PerCP-Cy5.5;PE-TexasRed (Red 613); Phloxin B (Magdala Red); Phorwite AR; PhorwiteBKL; Phorwite Rev; Phorwite RPA; Phosphine 3R; PhotoResist;Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH26; PKH67; PMIA;Pontochrome Blue Black; POPO-1; POPO-3; PO-PRO-1; PO-PRO-3; Primuline;Procion Yellow; Propidium Iodid (PI); PyMPO; Pyrene; Pyronine; PyronineB; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Resorufin;RH 414; Rhod-2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5GLD; Rhodamine 6G; Rhodamine B 540; Rhodamine B 200; Rhodamine B extra;Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine;Rhodamine Phalloidine; Rhodamine Red; Rhodamine WT; Rose Bengal;R-phycoerythrin (PE); red shifted GFP (rsGFP, S65T); S65A; S65C; S65L;S65T; Sapphire GFP; Serotonin; Sevron Brilliant Red 2B; Sevron BrilliantRed 4G; Sevron Brilliant Red B; Sevron Orange; Sevron Yellow L; sgBFP™;sgBFP™ (super glow BFP); sgGFP™; sgGFP™ (super glow GFP); SITS; SITS(Primuline); SITS (Stilbene Isothiosulphonic Acid); SPQ(6-methoxy-N-(3-sulfopropyl)-quinolinium); Stilbene; Sulphorhodamine Bcan C; Sulphorhodamine G Extra; Tetracycline; Tetramethylrhodamine;Texas Red™; Texas Red-X™ conjugate; Thiadicarbocyanine (DiSC3); ThiazineRed R; Thiazole Orange; Thioflavin 5; Thioflavin S; Thioflavin TCN;Thiolyte; Thiozole Orange; Tinopol CBS (Calcofluor White); TMR;TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; TriColor (PE-Cy5); TRITC(TetramethylRodaminelsoThioCyanate); True Blue; TruRed; Ultralite;Uranine B; Uvitex SFC; wt GFP; WW 781; XL665; X-Rhodamine; XRITC; XyleneOrange; Y66F; Y66H; Y66W; Yellow GFP; YFP; YO-PRO-1; YO-PRO-3; YOYO-1;and YOYO-3. Many suitable forms of these fluorescent compounds areavailable and can be used.

Other exemplary detectable labels include luminescent and bioluminescentmarkers (e.g., biotin, luciferase (e.g., bacterial, firefly, clickbeetle and the like), luciferin, and aequorin), radiolabels (e.g., 3H,125I, 35S, 14C, or 32P), enzymes (e.g., galactosidases, glucorinidases,phosphatases (e.g., alkaline phosphatase), peroxidases (e.g.,horseradish peroxidase), and cholinesterases), and calorimetric labelssuch as colloidal gold or colored glass or plastic (e.g., polystyrene,polypropylene, and latex) beads. Patents teaching the use of such labelsinclude U.S. Pat. Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345,4,277,437, 4,275,149, and 4,366,241, each of which is incorporatedherein by reference.

Means of detecting such labels are well known to those of skill in theart. Thus, for example, radiolabels can be detected using photographicfilm or scintillation counters, fluorescent markers can be detectedusing a photo-detector to detect emitted light. Enzymatic labels aretypically detected by providing the enzyme with an enzyme substrate anddetecting the reaction product produced by the action of the enzyme onthe enzyme substrate, and calorimetric labels can be detected byvisualizing the colored label.

In some embodiments, the detectable label is a fluorophore or a quantumdot. Without wishing to be bound by a theory, using a fluorescentreagent can reduce signal-to-noise in the imaging/readout, thusmaintaining sensitivity. Accordingly, in some embodiments, prior todetection, the microbes isolated from or remained bound on themicrobe-targeting substrate can be stained with at least one stain,e.g., at least one fluorescent staining reagent comprising amicrobe-binding molecule, wherein the microbe-binding molecule comprisesa fluorophore or a quantum dot. Examples of fluorescent stains include,but are not limited to, any microbe-targeting element (e.g.,microbe-specific antibodies or any microbe-binding proteins or peptidesor oligonucleotides) typically conjugated with a fluorophore or quantumdot, and any fluorescent stains used for detection as described herein.

In some embodiments, a labeling molecule can be configured to include a“smart label”, which is undetectable when conjugated to themicrobe-binding molecules, but produces a color change when releasedfrom the engineered molecules in the presence of a microbe enzyme. Thus,when a microbe binds to the engineered microbe-binding molecules, themicrobe releases enzymes that release the detectable label from theengineered molecules. An observation of a color change indicatespresence of the microbe in the sample.

In some embodiments, the microbe-targeting substrate can be conjugatedwith a label, such as a detectable label or a biotin label.

In some embodiments, the labeling molecule can comprise MBL or amicrobe-binding molecule described herein. In one embodiment, thelabeling molecule comprises FcMBL. Without wishing to be bound by atheory, labeling molecules based on MBL, and FcMBL in particular, attachselectively to a broad range of microbes, and so they enable the methoddescribed herein to detect the majority of blood-borne microbes withhigh sensitivity and specificity.

Any method known in the art for detecting the particular label can beused for detection. Exemplary methods include, but are not limited to,spectrometry, fluorometry, microscopy imaging, immunoassay, and thelike. While the microbe capture step can specifically capture microbes,it can be beneficial to use a labeling molecule that can enhance thisspecificity. If imaging, e.g., microscopic imaging, is to be used fordetecting the label, the staining can be done either prior to or afterthe microbes have been laid out for microscopic imaging. Additionally,imaging analysis can be performed via automated image acquisition andanalysis.

For optical detection, including fluorescent detection, more than onestain or dye can be used to enhance the detection or identification ofthe microbe. For example, a first dye or stain can be used that can bindwith a genus of microbes, and a second dye or strain can be used thatcan bind with a specific microbe. Colocalization of the two dyes thenprovides enhanced detection or identification of the microbe by reducingfalse positive detection of microbes.

In some embodiments, microscopic imaging can be used to detect signalsfrom label on the labeling agent. Generally, the microbes in thesubsample are stained with a staining reagent and one or more imagestaken from which an artisan can easily count the number of cells presentin a field of view.

In particular embodiments, microbe can be detected through use of one ormore enzyme assays, e.g., enzyme-linked assay (ELISA). Numerous enzymeassays can be used to provide for detection. Examples of such enzymeassays include, but are not limited to, beta-galactosidase assays,peroxidase assays, catalase assays, alkaline phosphatase assays, and thelike. In some embodiments, enzyme assays can be configured such that anenzyme will catalyze a reaction involving an enzyme substrate thatproduces a fluorescent product. Enzymes and fluorescent enzymesubstrates are known and are commercially available (e.g.,Sigma-Aldrich, St. Louis, Mo.). In some embodiments, enzyme assays canbe configured as binding assays that provide for detection of microbe.For example, in some embodiments, a labeling molecule can be conjugatedwith an enzyme for use in the enzyme assay. An enzyme substrate can thenbe introduced to the one or more immobilized enzymes such that theenzymes are able to catalyze a reaction involving the enzyme substrateto produce a detectable signal.

In some embodiments, an enzyme-linked assay (ELISA) can be used todetect signals from the labeling molecule. In ELISA, the labelingmolecule can comprise an enzyme as the detectable label. Each labelingmolecule can comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or more) enzymes. Additionally, each labeling molecule can comprise oneor more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) sites for bindingwith a microbe. Without wishing to be bound by a theory, presence ofmultimeric probe molecules can enhance ELISA signal.

For ELISA, any labeling molecule conjugated to an enzyme can be used.Exemplary labeling molecule include those comprising MBL, FcMBL,AKT-FcMBL, wheat germ agglutinin, lectins, antibodies (e.g.,gram-negative antibodies or gram-positive antibodies), antigen bindingfragments of antibodies, aptamers, ligands (agonists or antagonists) ofcell-surface receptors and the like.

In some embodiments, the labeling molecule can comprise MBL or FcMBLlabeled with a detectable label.

Similarly, a variety of enzymes can be used, with either colorimetric orfluorogenic substrates. In some embodiments, the reporter-enzymeproduces a calorimetric change which can be measured as light absorptionat a particular wavelength. Exemplary enzymes include, but are notlimited to, beta-galactosidases, peroxidases, catalases, alkalinephosphatases, and the like.

In some embodiments, the enzyme is a horseradish peroxidase (HRP).

In some embodiments, the enzyme is an alkaline peroxidase (AP).

A microbe-binding molecule and the enzyme can be linked to each other bya linker. In some embodiments, the linker between the microbe-bindingmolecule and the enzyme is an amide bond. In some embodiments, thelinker between the microbe-binding molecule and the enzyme is adisulfide (S—S) bond.

When the microbe-binding molecule is a peptide, polypeptide or aprotein, the enzyme can be linked at the N-terminus, the C-terminus, orat an internal position of the microbe-binding molecule. Similarly, theenzyme can be linked by its N-terminus, C-terminus, or an internalposition.

In one embodiment, the ELISA probe molecule can comprise a MBL or aportion there of or a FcMBL molecule linked to a HRP. Conjugation of HRPto any proteins and antibodies are known in the art. In one embodiment,FcMBL-HRP construct is generated by direct coupling HRP to FcMBL usingany commercially-available HRP conjugation kit. In some embodiments, themicrobes isolated from or remained bound on the microbe-targetingsubstrate can be incubated with the HRP-labeled microbe-bindingmolecules, e.g., MBL or a portion thereof, or a FcMBL molecule linked toa HRP for a period of time, e.g., at least about 5 mins, at least about10 mins, at least about 15 mins, at least about 20 mins, at least about25 mins, at least about 30 mins. The typical concentrations ofHRP-labeled molecules used in the ELISA assay can range from about 1:500to about 1:20,000 dilutions. In one embodiment, the concentration ofHRP-labeled microbe-binding molecules, e.g., MBL or a portion thereof,or a FcMBL molecule linked to a HRP molecule, can be about 1:1000 toabout 1:10000 dilutions.

In one embodiment, the ELISA probe molecule can comprise a MBL or aportion thereof, or a FcMBL molecule linked to a AP. Conjugation of APto any proteins and antibodies are known in the art. In one embodiment,FcMBL-AP construct is generated by direct coupling AP to FcMBL using anycommercially-available AP conjugation kit. In some embodiments, themicrobes isolated from or remained bound on the microbe-targetingsubstrate can be incubated with the AP-labeled microbe-binding molecule,e.g., MBL or a portion thereof, or a FcMBL molecule linked to a AP for aperiod of time, e.g., at least about 5 mins, at least about 10 mins, atleast about 15 mins, at least about 20 mins, at least about 25 mins, atleast about 30 mins. The typical concentrations of AP-labeled moleculesused in the ELISA assay can range from about 1:1000 to about 1:20,000dilutions. In one embodiment, the concentration of AP-labeledmicrobe-binding molecules, e.g., MBL or a portion thereof, or a FcMBLmolecule linked to a AP molecule, can be about 1:5000 to about 1:10000dilutions.

Following incubation with the ELISA probe molecules, the sample can bewashed with a wash buffer one or more (e.g., 1, 2, 3, 4, 5 or more)times to remove any unbound probes. An appropriate substrate for theenzyme (e.g., HRP or AP) can be added to develop the assay. Chromogenicsubstrates for the enzymes (e.g., HRP or AP) are known to one of skillin the art. A skilled artisan can select appropriate chromogenicsubstrates for the enzyme, e.g., TMB substrate for the HRP enzyme, orBCIP/NBT for the AP enzyme. In some embodiments, the wash buffer usedafter incubation with an ELISA probe molecule can contain calcium ionsat a concentration of about at least about 0.01 mM, at least about 0.05mM, at least about 0.1 mM, at least about 0.5 mM, at least about 1 mM,at least about 2.5 mM, at least about 5 mM, at least about 10 mM, atleast about 20 mM, at least about 30 mM, at least about 40 mM, at leastabout 50 mM or more. In alternative embodiments, the wash buffer usedafter incubation with an ELISA probe molecule can contain no calciumions. In some embodiments, the wash buffer used after incubation with anELISA probe molecule can contain a chelating agent. A wash buffer can beany art-recognized buffer used for washing between incubations withantibodies and/or labeling molecules. An exemplary wash buffer caninclude, but is not limited to, TBST.

In some embodiments, without wishing to be bound by theory, it can bedesirable to use a wash buffer without a surfactant or a detergent forthe last wash before addition of a chromogenic substrate, because asurfactant or detergent may have adverse effect to the enzymaticreaction with a chromogenic substrate.

One advantage of the ELISA-based approach is that the solid substratedoes not need to be dispersed or dissociated from the microbe beforebinding the secondary reagents. This is in contrast to microscopictechniques, in which excess residual solid substrate may obscure themicrobe during imaging. Furthermore, the optical readout components forELISA are likely cheaper than in the microscopy case, and there is noneed for focusing or for demanding that the sample be on the same focalplane. A further advantage of the ELISA-based approach is that it cantake advantage of commercially available laboratory equipment. Inparticular, when the solid substrate is magnetic, magnetic separationcan be automated using the KINGFISHER® system, the brief culture can beperformed using an airlift fermenter, and the colorimetric/fluorescentreadout can be attained using a standard plate reader.

Further amplification of the ELISA signal can be obtained bymultimerizing the recognition molecule (e.g., the microbe-bindingmolecule) or by multimerizing the detection enzyme (HRP, etc.). Forinstance, phage expression can be used to yield multimerized MBL andprovide a scaffold to increase the concentration of HRP (either throughdirect coupling of HRP to the phage particles or using an HRP-antiM13conjugated antibody).

In some embodiments, microbe can be detected through use of immunoassay.Numerous types of detection methods may be used in combination withimmunoassay based methods.

Without limitations, detection of microbes in a sample can also becarried out using light microscopy with phase contrast imaging based onthe characteristic size (5 um diameter), shape (spherical to elliptical)and refractile characteristics of target components such as microbesthat are distinct from all normal blood cells. Greater specificity canbe obtained using optical imaging with fluorescent or cytochemicalstains that are specific for all microbes or specific subclasses (e.g.calcofluor (1 μM to 100 μM) for chitin in fungi, fluorescent antibodiesdirected against fungal surface molecules, gram stains, acid-faststains, fluorescent MBL, fluorescent Fc-MBL, etc.).

Microbe detection can also be carried out using an epifluorescentmicroscope to identify the characteristic size (5 um diameter), shape(spherical to elliptical) and staining characteristics of microbes. Forexample, fungi stain differently from all normal blood cells, stronglybinding calcofluor (1 μM to 100 μM) and having a rigid ellipsoid shapenot found in any other normal blood cells.

In some embodiments, a microbe can be detected through use ofspectroscopy. Numerous types of spectroscopic methods can be used.Examples of such methods include, but are not limited to, ultravioletspectroscopy, visible light spectroscopy, infrared spectroscopy, x-rayspectroscopy, fluorescence spectroscopy, mass spectroscopy, plasmonresonance (e.g., Cherif et al., Clinical Chemistry, 52:255-262 (2006)and U.S. Pat. No. 7,030,989; herein incorporated by reference), nuclearmagnetic resonance spectroscopy, Raman spectroscopy, fluorescencequenching, fluorescence resonance energy transfer, intrinsicfluorescence, ligand fluorescence, and the like.

In some embodiments, a microbe can be detected through use offluorescence anisotropy. Fluorescence anisotropy is based on measuringthe steady state polarization of sample fluorescence imaged in aconfocal arrangement. A linearly polarized laser excitation sourcepreferentially excites fluorescent target molecules with transitionmoments aligned parallel to the incident polarization vector. Theresultant fluorescence is collected and directed into two channels thatmeasure the intensity of the fluorescence polarized both parallel andperpendicular to that of the excitation beam. With these twomeasurements, the fluorescence anisotropy, r, can be determined from theequation: r=(Intensity parallel−Intensity perpendicular)/(Intensityparallel+2(Intensity perpendicular)) where the I terms indicateintensity measurements parallel and perpendicular to the incidentpolarization. Fluorescence anisotropy detection of fluorescent moleculeshas been described. Accordingly, fluorescence anisotropy can be coupledto numerous fluorescent labels as have been described herein and as havebeen described in the art.

In some embodiments, microbe can be detected through use of fluorescenceresonance energy transfer (FRET). Fluorescence resonance energy transferrefers to an energy transfer mechanism between two fluorescentmolecules. A fluorescent donor is excited at its fluorescence excitationwavelength. This excited state is then nonradiatively transferred to asecond molecule, the fluorescent acceptor. Fluorescence resonance energytransfer may be used within numerous configurations to detect capturedmicrobe. For example, in some embodiments, a first labeling molecule canbe labeled with a fluorescent donor and second labeling molecule can belabeled with a fluorescent acceptor. Accordingly, such labeled first andsecond labeling molecules can be used within competition assays todetect the presence and/or concentration of microbe in a sample.Numerous combinations of fluorescent donors and fluorescent acceptorscan be used for detection.

In some embodiments, a microbe can be detected through use ofpolynucleotide analysis. Examples of such methods include, but are notlimited to, those based on polynucleotide hybridization, polynucleotideligation, polynucleotide amplification, polynucleotide degradation, andthe like. Methods that utilize intercalation dyes, fluorescenceresonance energy transfer, capacitive deoxyribonucleic acid detection,and nucleic acid amplification have been described, for example, in U.S.Pat. No. 7,118,910 and No. 6,960,437; herein incorporated by reference).Such methods can be adapted to provide for detection of one or moremicrobe nucleic acids. In some embodiments, fluorescence quenching,molecular beacons, electron transfer, electrical conductivity, and thelike can be used to analyze polynucleotide interaction. Such methods areknown and have been described, for example, in Jarvius, DNA Tools andMicrofluidic Systems for Molecular Analysis, Digital ComprehensiveSummaries of Uppsala Dissertations from the Faculty of Medicine 161,ACTA UNIVERSITATIS UPSALIENSIS UPPSALA 2006, ISBN: 91-554-6616-8;Singh-Zocchi et al, Proc. Natl. Acad. Sci, 100:7605-7610 (2003); Wang etal. Anal. Chem, 75:3941-3945 (2003); and Fan et al, Proc. Natl. Acad.Sci, 100:9134-9137 (2003) and in U.S. Pat. No. 6,958,216; No. 5,093,268;and 6,090,545, the content of all of which is incorporated herein byreference. In some embodiments, the polynucleotide analysis is bypolymerase chain reaction (PCR). The fundamentals of PCR are well-knownto the skilled artisan, see, e.g. McPherson, et al., PCR, A PracticalApproach, IRL Press, Oxford, Eng. (1991), hereby incorporated byreference.

In some embodiments, a metabolic assay is used to determine the relativenumber of microbes in a sample compared to a control. As will beapparent to one of ordinary skill in the art any metabolic indicatorthat can be associated with cells can be used, such as but not limitedto, turbidity, fluorescent dyes, and redox indicators such as, but notlimited to, Alamar Blue, MTT, XTT, MTS, and WST. Metabolic indicatorscan be components inherent to the cells or components added to theenvironment of the cells. In some embodiments, changes in or the stateof the metabolic indicator can result in alteration of ability of themedia containing the sample to absorb or reflect particular wavelengthsof radiation.

Exemplary metabolic assays include, but are not limited to, ATPLuminescence, reactive oxygen species (ROS) assays, Resazurin assays,Luminol, MTT-metabolic assays, and the like. Further, as one of skill inthe art is well aware, kits and methods for carrying out metabolicassays are commercially available. For example,2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (2-NBDG),ATP Determination Kit, AMPLEX® Red Galactose/Galactose Oxidase AssayKit, AMPLEX® Red Glucose/Glucose Oxidase Assay Kit, AMPLEX® Red GlutamicAcid/Glutamate Oxidase Assay Kit, AMPLEX® Red HydrogenPeroxide/Peroxidase Assay Kit, AMPLEX® Red Monoamine Oxidase Assay Kit,AMPLEX® Red Neuraminidase (Sialidase) Assay Kit, AMPLEX® RedPhosphatidylcholine-Specific Phospholipase C Assay Kit, AMPLEX® RedSphingomyelinase Assay kit, AMPLEX® Red Uric Acid/Uricase Assay Kit,AMPLEX® Red Xanthine/Xanthine Oxidase Assay Kit, THIOLTRACKER™ Violet(Glutathione Detection Reagent), THIOLTRACKER™ Violet (GlutathioneDetection Reagent), and VYBRANT® Cell Metabolic Assay Kit fromInvitrogen; Adenosine 5′-triphosphate (ATP) Luminescence Assay Kit(ENLITEN® from Promega; ATPLITE™ from PerkinElmer Life Sciences; ATPBioluminescence Assay kit HS II from Boehringer Mannheim, Germany;Adenosine 5′-triphosphate (ATP) Luminescence Assay Kit from EMDMillipore; Reactive Oxygen Species (ROS) Assays from Cell BioLabs, Inc.;Cellular Reactive Oxygen Species Detection Assay Kit from ABCAM®; hROSDetection Kit from Cell Technology, Inc.; and ABTS Antioxidant AssayKit, ORAC Antioxidant Assay Kit, OxiSelect HORAC Activity Assay Kit,OxiSelect In vitro ROS/RNS Assay Kit (Green Fluorescence), OxiSelectIntracellular ROS Assay Kit (Green Fluorescence), OxiSelect ORACActivity Assay Kit, OxiSelect Total Antioxidant Capacity (TAC) AssayKit, and Total Antioxidant Capacity Assay Kit from BioCat.

In some embodiments, microbes isolated from or remained bound onmicrobe-targeting substrate can be labeled with nucleic acid barcodesfor subsequent detection and/or multiplexing detection. Nucleic acidbarcoding methods for detection of one or more analytes in a sample arewell known in the art.

In other embodiments, the captured microbe can be analyzed and/ordetected in the capture chamber or capture and visualization chamber ofa rapid microbe diagnostic device described in the Int. Pat. App. No.Int. Pat. App. No. WO 2011/091037, filed Jan. 19, 2011. Alternatively,the captured microbe can be recovered (i.e., removed) and analyzedand/or detected.

In some embodiments, the captured microbe is recovered and analyzedand/or detected using a particle on membrane assay as described in U.S.Pat. No. 7,781,226, content of which is incorporated herein byreference. A particle on membrane assay as described in U.S. Pat. No.7,781,226 can be operably linked with a rapid microbe diagnostic deviceof the Int. Pat. App. No. Int. Pat. App. No. WO 2011/091037 to reducethe number of sample handling steps, automate the process and/orintegrate the capture, separation and analysis/detection steps into amicrofluidic device.

In some embodiments, microbe capture, separation and analysis can bedone using a hybrid microfluidic SPR and molecular imagining device asdescribed in U.S. Pat. App. Pub. No. US 2011/0039280.

In some embodiments, the processes or assays described herein can detectthe presence or absence of a microbe and/or identify a microbe in a testsample in less than 24 hours, less than 12 hours, less than 10 hours,less than 8 hours, less than 6 hours, less than 4 hours, less than 3hours, less than 2 hours, less than 1 hour, or lower. In someembodiments, the processes or assays described herein can detect thepresence or absence of a microbe and/or identify a microbe in a testsample in less than 6 hours, less than 4 hours, less than 3 hours, lessthan 2 hours, less than 1 hour, or lower.

Optional Additional Analyses or Treatment—Culturing:

In some embodiments of any aspects described herein, the assay orprocess can further comprise culturing any microbe bound on themicrobe-targeting substrate (e.g., microbe-targeting magneticmicrobeads) for a period of time. In such embodiments, the microbe boundon the microbe-targeting substrate can expand in population by at leastabout 10% after culturing for a period of time.

In some embodiments, the microbe bound on the microbe-targetingsubstrate (e.g., microbe-targeting magnetic microbeads) can be culturedfor a period of time, e.g., at least about 15 mins, at least about 30mins, at least about 1 hour, at least about 2 hours, at least about 3hours, at least about 6 hours, at least about 9 hours, at least about 12hours, at least about 18 hours, at least about 24 hours or longer. Insome embodiments, the microbe bound on the microbe-targeting substrate(e.g., microbe-targeting magnetic microbeads) can be cultured for atleast about 30 mins to at least about 3 hours.

In some embodiments, the number of microbes bound on themicrobe-targeting substrate (e.g., microbe-targeting magneticmicrobeads) after culturing for a certain period of time can beincreased or expanded by at least about 30%, at least about 40%, atleast about 50%, at least about 60%, at least about 70%, at least about80%, at least about 90%, at least about 100%, as compared to the numberof the microbes originally bound on the microbe-targeting substrate. Insome embodiments, the number of microbes bound on the microbe-targetingsubstrate (e.g., microbe-targeting magnetic microbeads) after culturingfor a certain period of time can be increased or expanded by at leastabout 1.5-fold, at least about 2-fold, at least about 3-fold, at leastabout 4-fold, at least about 5-fold, at least about 10-fold, at leastabout 50-fold, at least about 100-fold, at least about 500-fold, atleast about 1000-fold, at least about 10000-fold, at least about100000-fold, as compared to the number of the microbes originally boundon the microbe-targeting substrate.

In some embodiments, the microbes bound on the microbe-targetingsubstrates (e.g., microbe-targeting magnetic microbeads) can be culturedon a microbe-compatible culture medium, e.g., plated on an agar plate orcultured in LB broth. One of skill in the art will readily recognizemicrobial culture techniques, including, but not limited to, the use ofincubators and/or equipment used to provide a gentle agitation, e.g.,rotator platforms, and shakers, if necessary, e.g., to prevent the cellsfrom aggregation without subjecting them to a significant shear stressand provide aerial agitation.

The microbes can remain bound on the microbe-targeting substrate (e.g.,microbe-targeting magnetic microbeads) during detection and/oradditional analyses described herein or they can be detached, eluted offor removed from a microbe-targeting substrate prior to detection oradditional analyses described herein. In some embodiments where thebound microbes are desired to be detached, eluted off or removed from amicrobe-targeting substrate, the microbe-binding molecules of themicrobe-targeting substrate can be further contacted with a low pHbuffer, e.g., a pH buffer less than 6, less than 5, less than 4, lessthan 3, less than 2, less than 1 or lower. In some embodiments, a low pHbuffer that does not cause precipitation of a chelating agent, ifpresent, can be used. In one embodiment, a low pH buffer can bearginine. In another embodiment, a low pH buffer can be pyrophosphate.

In some embodiments of any aspects described herein, the microbe-bindingmolecules of the microbe-targeting substrate can be further contactedwith a low pH buffer and a chelating agent. In some embodiments, thecontact of the microbe-binding molecules of the microbe-targetingsubstrate with the low pH buffer and the chelating agent can beconcurrent or sequentially. In one embodiment, the microbe-bindingmolecules of the microbe-targeting substrate can be further contactedwith arginine (e.g., 2 M) with EDTA or EGTA at pH 4.4.

The isolated microbes can then be used for analyses described earlier oradditional treatment, e.g., expansion in culture, antibiotic sensitivitytesting, sequencing and/or DNA or RNA analysis.

Optional Additional Analyses or Treatment-Antibiotic Sensitivity orSusceptibility Testing:

In some embodiments of any aspects described herein, the process orassay described herein can further comprise subjecting the microbesbound on the microbe-targeting substrate (e.g., microbe-targetingmagnetic microbeads) and/or the expanded cultures of microbes isolatedfrom the microbe-targeting substrate (e.g., microbe-targeting magneticmicrobeads) to one or more antibiotics. The response of the microbe toan antibiotic can then be evaluated with any known methods in the art,e.g., by measuring the viability of microbes. Thus, an appropriateantibiotic can be identified for treatment of an infection caused by amicrobe, even though the specific species of the microbe bound onto themicrobe-targeting substrate is initially unknown. Additional details foruse of engineered microbe-targeting molecules described herein inantibiotic sensitivity testings can be found, e.g., in U.S. Prov. App.Nos. 61/604,878 filed Feb. 29, 2012 and 61/647,860 filed May 16, 2012.

Any processes or steps described herein can be performed by a module ordevice. While these are discussed as discrete processes, one or more ofthe processes or steps described herein can be combined into one systemfor carrying out the assays of any aspects described herein.

Exemplary Embodiments of Methods for Diagnosing or Locating a MicrobialInfection or Contamination

In general, embodiments of the assays or processes of any aspectsdescribed herein can be used to detect the presence or absence of amicrobe and/or microbial matter in a test sample or in situ (e.g., wherethe microbe actually resides, e.g., in a water reservoir or on a workingsurface). For example, in some embodiments, a test sample, e.g.,obtained from a subject or an environmental source, or an environmentalsurface can be contacted with engineered microbe-binding molecules orengineered microbe-binding substrates described herein, such that anymicrobes, if present, in the test sample or environmental surface can becaptured by the engineered microbe-binding molecules or engineeredmicrobe-binding substrates e.g., using any embodiments of the exemplaryprocess described above. In some embodiments, the captured microbesbound on the engineered microbe-binding molecules and/or microbe-bindingsubstrates can then be subjected to different analyses as describedabove, e.g., for identifying a microbe genus or species such as byimmunoassay (e.g., using antibodies to a specific microbe), massspectrometry, PCR, etc. In alternative embodiments where the engineeredmicrobe-binding molecules comprise an imaging agent (e.g., a bubble, aliposome, a sphere, a diagnostic contrast agent or a detectable labeldescribed herein), the binding of the microbes to the engineeredmicrobe-binding molecules can be detected in situ for identification oflocalized microbial infection or contamination, and also allow localizedtreatment of the infection or contamination.

In some embodiments, the assays or processes described herein can beused to diagnose or locate a microbial infection in situ in a subject.For example, engineered microbe-targeting microbeads comprising animaging agent (e.g., the engineered microbe-targeting microbeads can belinked to an imaging agent, e.g., a bubble, a liposome, a sphere, adiagnostic contrast agent or a detectable label described herein) can beadministered to a subject, either systemically (e.g., by injection), orlocally. In such embodiments, the engineered microbe-targetingmicrobeads comprising an imaging agent can be used to identify and/orlocalize pockets of localized microbial infection (e.g., in a tissue) inthe subject and optionally allow localized treatment of the microbialinfection, which is described in the section “Exemplary Compositions andMethods for Treating and/or Preventing a Microbial Infection” below.

While an engineered microbe-binding molecule described herein (e.g.,FcMBL) can bind to a broad spectrum of microbes, in certain embodiments,a microbe species (e.g., S. aureus) can be isolated and/ordifferentiated from another species (E. coli) based on their distinctabilities of binding to the engineered microbe-binding molecules orsubstrates described herein. For example, the inventors havedemonstrated that S. aureus can bind to FcMBL via both calcium-dependentMBL-mediated interaction and calcium-independent Fc-mediatedinteraction, while E. coli can bind to FcMBL primarily viacalcium-dependent MBL-mediated interaction. Without wishing to belimiting, an exemplary method for diagnosing an infection caused by S.aureus based on such unique ability of S. aureus binding to anengineered microbe-binding molecule (e.g., FcMBL) is described below forillustration purposes. One of skill in the art can readily make anynecessary modifications to the exemplary illustration and/or adopt anyembodiments of the assays or processes described herein to detect thepresence or absence of any microbe in a test sample or in situ and/ordiagnosing different kinds of microbial infections in a subject.

For example, there is a strong need for more rapid and/or effectivediagnostic methods for distinguishing at least S. aureus from otherbacteria, e.g., E. coli, which can permit physicians to initiate anappropriate drug therapy early on, rather than starting with asub-optimal or a completely ineffective antibiotic. A delay in treatmentof a microbial infection, e.g., S. aureus, can significantly affect thetreatment outcome, and can be sometimes fatal.

Accordingly, in some embodiments, the assays or processes describedherein can be used to distinguish a protein A-expressing microbe or aprotein G-expressing microbe from a protein A- and protein G-negativemicrobe (e.g., E. coli) in a test sample. In particular, the inventorshave demonstrated that S. aureus can be differentiated from E. coliusing some embodiments of the assays or processes described herein. Insome embodiments, a microbe-targeting substrate comprises a substratecoupled to a fusion protein between the Fc portion of human IgG1 and theneck and carbohydrate recognition domain (CRD) of human Mannose BindingLectin (MBL) can be used for such microbial differentiation.

Accordingly, exemplary methods of determining the presence or absence ofStaphylococcus aureus infection in a subject are also provided herein.For example, the method can comprise contacting at least a first volumeof a test sample with a microbe-targeting substrate described herein inthe presence of a chelating agent. Alternatively, the method cancomprise (i) contacting at least a first volume of a test sample with amicrobe-targeting substrate described herein in the presence of freecalcium ions, and (ii) contacting the microbe-binding molecule of themicrobe-targeting substrate described herein, upon the contact with thetest sample, with a solution comprising a chelating agent. In someembodiments described herein, the method can further comprise analyzingthe microbe-targeting substrate for the presence or absence of a boundmicrobe. The presence of a microbe bound onto the microbe-targetingsubstrate indicates the presence of a protein-A expressing microbe or aprotein G-expressing microbe in the test sample; and the absence of amicrobe bound onto the microbe-targeting substrate indicates the absenceof a protein-A expressing or a protein G-expressing microbe in the testsample.

In some embodiments, the method can further comprise administering orprescribing to the subject an antimicrobial agent when the subject isdetected with S. aureus. Non-limiting examples of an antimicrobial agentcan include any therapeutic agent for treatment of S. aureus. In someembodiments, an antimicrobial agent can be an antibiotic commonlyindicated for treatment of S. aureus, including, but not limited to,penicillin, methicillin, nafcillin, oxacillin, cloxacillin,dicloxacillin, flucloxacillin, vancomycin, and any combinations thereof.

In some embodiments where a microbe is absent on the microbe-targetingsubstrate, the method can further comprise analyzing the test sample orthe solution comprising the chelating agent after removal of themicrobe-targeting substrate to determine the presence or absence of aprotein A- and protein G-negative microbe. For example, additionalcalcium ions (e.g., calcium salts) can be added to the test sample orthe solution comprising the chelating agent in an amount more than whatis needed to react with substantially all of the chelating agentmolecules such that there are free calcium ions available to mediatecarbohydrate recognition domain (e.g., MBL)-mediated binding between amicrobe and the microbe-targeting substrate. A fresh microbe-targetingsubstrate can then be contacted with the treated test sample or thesolution comprising the chelating agent in the presence of free calciumions to detect the presence or absence of a protein A- and proteinG-negative microbe (e.g., E. coli). Alternatively, a freshmicrobe-targeting substrate can be contacted with a fresh volume of thetest sample in the presence of free calcium ions (e.g., addition of acalcium salt at a concentration, e.g., of at least about 1 mM, at leastabout 5 mM, or higher) to detect the presence or absence of a protein A-and protein G-negative microbe (e.g., E. coli). Detection methodsdescribed above for a protein A-expressing or protein G-expressingmicrobe bound on a microbe-targeting substrate can be used for suchpurposes as well. Detection methods described in the InternationalApplication No. WO 2011/090954, the content of which is incorporatedherein by reference, can also be employed herein to determine thepresence or absence of protein A- and protein G-negative microbes (e.g.,E. coli).

In those embodiments, when a microbe (e.g., protein A- and proteinG-negative microbe (e.g., E. coli)) is detected in a subject, the methodcan further comprise administering or prescribing to the subject anappropriate antimicrobial agent described herein to treat thecorresponding microbe (e.g., the protein A- and protein G-negativemicrobe, e.g., E. coli).

Without wishing to be bound by theory, some embodiments of theengineered microbe-binding molecules can be used to opsonize a microbe,which is then cleared out by an innate immune response. In someembodiments, FcMBL protein can be a more potent opsonin of a microbe,g., S. aureus than Fc or wild-type MBL. Accordingly, in someembodiments, when the subject is diagnosed with a microbial infectionusing the methods described herein, the subject can be administered orprescribed with a composition comprising at least one engineeredmicrobe-binding molecule described herein.

Without limitations, the methods of any aspects described herein can beused to diagnose a microbe that is resistant to at least one, at leasttwo, at least three, at least four or more antibiotics. For example, inone embodiment, the methods described herein can be used to diagnosemethicillin-resistant S. aureus. In another embodiment, the methodsdescribed herein can be used to diagnose vancomycin-resistant S. aureus.

Exemplary Compositions and Methods for Treating and/or Preventing aMicrobial Infection

The binding of microbes to engineered microbe-targeting molecules canfacilitate isolation and removal of microbes and/or microbial matterfrom an infected area. Accordingly, another aspect provided hereinrelate to compositions for treating and/or preventing a microbialinfection or microbial contamination comprising one or more engineeredmicrobe-targeting molecules or microbe-targeting substrates (e.g.,microbe-targeting magnetic microbeads) described herein.

In some embodiments, the composition can be formulated for treatingand/or preventing a microbial infection or a microbial contaminationpresent in an environmental surface. The term “environmental surface” asused herein refers to any surface and/or body of an environment or anobject. The environmental object can be a non-living object or a livingobject, e.g., a botanical plant. Examples of an environmental surfacecan include, but is not limited to, a medical device, an implantabledevice, a surface in a hospital or clinic (e.g., an operating room or anintensive-care unit), a machine or working surface for manufacturing orprocessing food or pharmaceutical products (e.g., drugs, therapeuticagents or imaging agents), a cell culture, a water treatment plant, awater reservoir and a botanical plant.

In some embodiments, the composition can be formulated for treatingand/or preventing microbial infection in a body fluid of a subject,e.g., blood. While in some embodiments, the engineered microbe-targetingmolecules of the composition described herein can capture microbesand/or microbial matter in a circulating body fluid, e.g., blood, inother embodiments, the engineered microbe-targeting molecules canopsonize a microbe and/or microbial matter such that the microbe and/ormicrobial matter can be recognized by an innate immune system forclearance.

Unlike wild-type MBL that can induce systemic complement activation(see, e.g., Sprong T. (2009) Clin Infect Dis. 49: 1380-1386), in someembodiments, the engineered microbe-targeting molecules can act asdominant negative molecules by binding microbes and/or microbial matterwithout stimulating downstream inflammatory cascades, and thus reducesystem inflammatory syndromes and/or sepsis symptoms in vivo, e.g.,reduction of disseminated intravascular coagulation (DIC).

Alternatively, the engineered microbe-targeting molecules can localize amicrobe and can thus prevent it from spreading, e.g., deeper into awound. In particular, the inventors have demonstrated that S. aureus canstrongly bind to some embodiments of the engineered microbe-targetingmolecules (e.g., microbe-binding magnetic microbeads) due to thepresence of both carbohydrate patterns and protein A on its microbialsurface capable of independent binding to the engineeredmicrobe-targeting molecules. Thus, in some embodiments, the engineeredmicrobe-targeting molecules can be used to localize a microbe load,which can then be easily removed from an infected area. In someembodiments, the microbead can be labeled for specific imaging ofinfected sites. For SPECT imaging the tracer radioisotopes typicallyused such as iodine-123, technetium-99m, xenon-133, thallium-201, andfluorine-18 can be used. Technetium 99m can be used for scintigraphicassay. Iodine-derived or other radioopaque contrast agents can also beincorporated in the beads for radiographic or CT-scan imaging. The useof paramagnetic or superparamagnetic microbeads can be used for magneticresonance imaging as contrast agents to alter the relaxation times ofatoms within a nidus of infection. In another embodiment, themicrospheres can be fluorescently dyed and applied to a surgical woundto determine the extension of an infectious process. This can be usefulfor assisting the surgeon in distinguishing between infected and healthytissues during debridment surgeries for osteomyelitis, cellulitis orfasciitis.

Accordingly, another aspect provided herein related to compositions fortreating and/or preventing a microbial infection in a tissue of asubject. In some embodiments, the composition comprises at least oneengineered microbe-targeting molecule as described herein. In someembodiments, the amount of the engineered microbe-targeting moleculesand/or microbe-targeting substrates present in the composition issufficient to reduce the growth and/or spread of the microbe in thetissue of the subject. The phrase “reducing the growth and/or spread ofthe microbe in the tissue” as used herein refers to reducing the numberof colonies of the microbe and/or movement of the microbe in the tissue.In some embodiments, the engineered microbe-targeting molecule cancapture and localize a microbe present in a tissue such that the numberof colonies of the microbe in the tissue can be reduced by at leastabout 30%, at least about 40%, at least about 50%, at least about 60%,at least about 70%, at least about 80%, at least about 90%, at leastabout 95%, at least about 98%, up to and including 100%, as compared toin the absence of the engineered microbe-targeting molecule. In someembodiments, the engineered microbe-targeting molecule can capture andlocalize a microbe present in a tissue such that the number of coloniesof the microbe in the tissue can be reduced by at least about 1.5-fold,at least about 2-fold, at least about 3-fold, at least about 4-fold, atleast about 5-fold, at least about 6-fold, at least about 7-fold, atleast about 8-fold, at least about 9-fold, at least about 10-fold, atleast about 15-fold, at least about 20-fold or more, as compared to inthe absence of the engineered microbe-targeting molecules. In oneembodiment, the binding of the engineered microbe-targeting moleculeswith a microbe (e.g., S. aureus) reduces the number of colonies by atleast about 4-fold to at least about 6-fold (e.g., at least about5-fold), as compared to in the absence of the engineeredmicrobe-targeting molecules, after a period of at least about 12 hours,at least about 16 hours or at least about 24 hours.

In other embodiments, the engineered microbe-targeting molecule cancapture and localize a microbe present in a tissue such that themovement of the microbe within the tissue (e.g., in terms of a distancetraveled deeper into the tissue and/or area of spread from the infectedsite) can be reduced by at least about 30%, at least about 40%, at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,at least about 90%, at least about 95%, at least about 98%, up to andincluding 100%, as compared to in the absence of the engineeredmicrobe-targeting molecule. In some embodiments, the engineeredmicrobe-targeting molecule can capture and localize a microbe present ina tissue such that the movement of the microbe within the tissue (e.g.,in terms of a distance traveled deeper into the tissue and/or area ofspread from the infected site) can be reduced by at least about1.5-fold, at least about 2-fold, at least about 3-fold, at least about4-fold, at least about 5-fold, at least about 6-fold, at least about7-fold, at least about 8-fold, at least about 9-fold, at least about10-fold, at least about 15-fold, at least about 20-fold or more, ascompared to in the absence of the engineered microbe-targeting molecule.

In some embodiments, the composition can further comprise at least oneof an antimicrobial agent and a drug delivery vehicle. For example, insome embodiments, the composition can further comprise at least 1, atleast 2, at least 3, at least 4, at least 5 or more antimicrobialagents. In some embodiments, the composition can further comprise one ora plurality of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60,70, 80, 90, 100, 500, 1000 or more) delivery vehicles. In someembodiments, the composition can further comprise a combination of atleast one (including at least 2, at least 3, at least 4, at least 5 ormore) antimicrobial agent and at least one (including 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 500, 1000 or more)drug delivery vehicle. As used herein, the term “drug delivery vehicle”generally refers to any material that can be used to carry an activeagent to a target site. Examples of drug delivery vehicles includes, butare not limited to, a cell, a peptide particle, a polymeric particle, adendrimer, a vesicle, a liposome, a hydrogel, a nucleic acid scaffold,an aptamer, and any combinations thereof,

In some embodiments where a drug delivery vehicle is included, anengineered microbe-targeting molecule and/or an antimicrobial agent canbe dispersed within (e.g., encapsulated or embedded in) a drug deliveryvehicle and/or coated on a surface of the drug delivery vehicle.

In some embodiments where the composition includes at least oneantimicrobial agent, the antimicrobial agent can be present as aseparate entity from the engineered microbe-targeting molecule and/or itcan be fused with at least one engineered microbe-targeting molecule,e.g., by genetic modification and/or chemical conjugation.

The term “antimicrobial agent” as used herein refers to any entity withantimicrobial activity, i.e. the ability to inhibit or reduce the growthand/or kill a microbe, e.g., by at least about 30%, at least about 40%,at least about 50%, at least about 75%, at least about 90% or more, ascompared to in the absence of an antimicrobial agent. An antimicrobialagent can be, for example, but not limited to, a silver nanoparticle, asmall molecule, a peptide, a peptidomimetics, an antibody or a fragmentthereof, a nucleic acid, an enzyme (e.g., an antimicrobialmetalloendopeptidase such as lysostaphin), an aptamer, a drug, anantibiotic, a chemical or any entity that can inhibit the growth and/orkill a microbe. Examples of an antimicrobial peptide that can beincluded in the composition described herein, include, but are notlimited to, mefloquine, venturicidin A, antimycin, myxothiazol,stigmatellin, diuron, iodoacetamide, potassium tellurite hydrate,aDL-vinylglycine, N-ethylmaleimide, L-allyglycine, diaryquinoline,betaine aldehyde chloride, acivcin, psicofuraine, buthioninesulfoximine, diaminopemelic acid, 4-phospho-D-erythronhydroxamic acid,motexafin gadolinium and/or xycitrin or modified versions or analoguesthereof.

In some embodiments, an antimicrobial agent included in the compositioncan be an antibiotic. As used herein, the term “antibiotic” is artrecognized and includes antimicrobial agents naturally produced bymicroorganisms such as bacteria (including Bacillus species),actinomycetes (including Streptomyces) or fungi that inhibit growth ofor destroy other microbes, or genetically-engineered thereof andisolated from such natural source. Substances of similar structure andmode of action can be synthesized chemically, or natural compounds canbe modified to produce semi-synthetic antibiotics. Exemplary classes ofantibiotics include, but are not limited to, (1) β-lactams, includingthe penicillins, cephalosporins monobactams, methicillin, andcarbapenems; (2) aminoglycosides, e.g., gentamicin, kanamycin, neomycin,tobramycin, netilmycin, paromomycin, and amikacin; (3) tetracyclines,e.g., doxycycline, minocycline, oxytetracycline, tetracycline, anddemeclocycline; (4) sulfonamides (e.g., mafenide, sulfacetamide,sulfadiazine and sulfasalazine) and trimethoprim; (5) quinolones, e.g.,ciprofloxacin, norfloxacin, and ofloxacin; (6) glycopeptides (e.g.,vancomycin, telavancin, teicoplanin); (7) macrolides, which include forexample, erythromycin, azithromycin, and clarithromycin; (8) carbapenems(e.g., ertapenem, doripenem, meropenem, and imipenem); (9)cephalosporins (e.g., cefadroxil, cefepime, and ceftobiprole); (10)lincosamides (e.g., clindamycin, and lincomycin); (11) monobactams(e.g., aztreonam); (12) nitrofurans (e.g., furazolidone, andnitrofurantoin); (13) Penicillins (e.g., amoxicillin, and Penicillin G);(14) polypeptides (e.g., bacitracin, colistin, and polymyxin B); and(15) other antibiotics, e.g., ansamycins, polymycins, carbacephem,chloramphenicol, lipopeptide, and drugs against mycobacteria (e.g., theones causing diseases in mammals, including tuberculosis (Mycobacteriumtuberculosis) and leprosy (Mycobacterium leprae), and any combinationsthereof.

Additional exemplary antimicrobial agent can include, but are notlimited to, antibacterial agents, antifungal agents, antiprotozoalagents, antiviral agents, and any mixtures thereof.

Exemplary antibacterial agents include, but are not limited to,Acrosoxacin, Amifioxacin, Amoxycillin, Ampicillin, Aspoxicillin,Azidocillin, Azithromycin, Aztreonam, Balofloxacin, lc Benzylpenicillin,Biapenem, Brodimoprim, Cefaclor, Cefadroxil, Cefatrizine, Cefcapene,Cefdinir, Cefetamet, Cefmetazole, Cefprozil, Cefroxadine, Ceftibuten,Cefuroxime, Cephalexin, Cephalonium, Cephaloridine, Cephamandole,Cephazolin, Cephradine, Chlorquinaldol, Chlortetracycline, Ciclacillin,Cinoxacin, Ciprofloxacin, Clarithromycin, Clavulanic Acid, Clindamycin,Clofazimine, Cloxacillin, Danofloxacin, Dapsone, Demeclocycline,Dicloxacillin, Difloxacin, Doxycycline, Enoxacin, Enrofloxacin,Erythromycin, Fleroxacin, Flomoxef, Flucloxacillin, Flumequine,Fosfomycin, Isoniazid, Levofloxacin, Mandelic Acid, Mecillinam,Metronidazole, Minocycline, Mupirocin, Nadifloxacin, Nalidixic Acid,Nifuirtoinol, Nitrofurantoin, Nitroxoline, Norfloxacin, Ofloxacin,Oxytetracycline, Panipenem, Pefloxacin, Phenoxymethylpenicillin,Pipemidic Acid, Piromidic Acid, Pivampicillin, Pivmecillinam,Prulifloxacin, Rufloxacin, Sparfloxacin, Sulbactam, Sulfabenzamide,Sulfacytine, Sulfametopyrazine, Sulphacetamide, Sulphadiazine,Sulphadimidine, Sulphamethizole, Sulphamethoxazole, Sulphanilamide,Sulphasomidine, Sulphathiazole, Temafioxacin, Tetracycline, Tetroxoprim,Tinidazole, Tosufloxacin, Trimethoprim, and pharmaceutically acceptablesalts or esters thereof.

Exemplary antifungal agents include, but are not limited to, Bifonazole,Butoconazole, Chlordantoin, Chlorphenesin, Ciclopirox Olamine,Clotrimazole, Eberconazole, Econazole, Fluconazole, Flutrimazole,Isoconazole, Itraconazole, Ketoconazole, Miconazole, Nifuroxime,Tioconazole, Terconazole, Undecenoic Acid, and pharmaceuticallyacceptable salts or esters thereof.

Exemplary antiprotozoal agents include, but are not limited to,Acetarsol, Azanidazole, Chloroquine, Metronidazole, Nifuratel,Nimorazole, Omidazole, Propenidazole, Secnidazole, Sineflngin,Tenonitrozole, Temidazole, Tinidazole, and pharmaceutically acceptablesalts or esters thereof.

Exemplary antiviral agents include, but are not limited to, Acyclovir,Brivudine, Cidofovir, Curcumin, Desciclovir, 1-Docosanol, Edoxudine, gQFameyclovir, Fiacitabine, Ibacitabine, Imiquimod, Lamivudine,Penciclovir, Valacyclovir, Valganciclovir, and pharmaceuticallyacceptable salts or esters thereof.

In some embodiments, the antimicrobial agent can include silver presentin any form, e.g., a nanoparticle, a colloid, a suspension, powder, andany combinations thereof.

In some embodiments, the composition can be used to treat and/or preventan infection caused by any microbe described herein. In one embodiment,the composition can be used to treat and/or prevent an infection causedby S. aureus.

In some embodiments, the composition can be used to treat and/or preventan infection caused by a microbe that is resistant to at least one, atleast two, at least three, at least four or more antimicrobial agentsdescribed herein. In one embodiment, the composition can be used totreat and/or prevent an infection caused by a microbe that is resistantto at least one, at least two, at least three, at least four or moreantibiotics described herein. For example, in one embodiment, thecomposition can be used to treat and/or prevent an infection caused bymethicillin-resistant S. aureus. In another embodiment, the compositioncan be used to treat and/or prevent an infection caused byvancomycin-resistant S. aureus.

Exemplary antimicrobial applications and/or products: The compositionsdescribed herein can be formulated or configured for differentapplications and/or products such antimicrobial products. In someembodiments, the composition described herein can be formulated aspharmaceutical compositions as described below, e.g., for therapeutictreatment as an antibiotic or antiseptic.

Wound Dressings:

In some embodiments, the composition described herein can be formulatedfor topical application, e.g., in wounds, lesions or abscesses. By wayof example only, in some embodiments, a plurality of engineeredmicrobe-targeting molecules can be blended with, attached to or coatedon a wound dressing, for example, but not limited to, a bandage, anadhesive, a gauze, a film, a gel, foam, hydrocolloid, alginate,hydrogel, paste (e.g., polysaccharide paste), a spray, a granule and abead.

In some embodiments, the wound dressing can include an additionalantimicrobial agent described herein and/or an antiseptic chemical,e.g., boracic lint and/or medicinal castor oil.

In one embodiment, a plurality of engineered microbe-targeting molecules(e.g., microbe-targeting microparticles or microbe-targeting magneticmicrobeads) can be attached or coated onto a wound dressing such as abandage or an adhesive. When such wound dressing is applied to a woundor a lesion, any microbe (e.g., S. aureus) and/or microbial matterpresent in the wound or lesion can bind and localized to the wounddressing. Thus, regular replacement of the wound dressing can remove themicrobe from the wound or lesion and thus prevent the microbe frommoving deeper into the wound or lesion for further infection.

In one embodiment, a plurality of engineered microbe-targeting molecules(e.g., microbe-targeting microparticles or microbe-targeting magneticmicrobeads) can be formulated into a wound dressing spray, which can behandy and used anywhere, e.g., during a transportation on an emergencyvehicle. When the wound dressing spray containing the microbe-targetingmagnetic microbeads, the microbe-targeting magnetic microbeads withbound microbes (e.g., S. aureus) can be removed from the wound with amagnetic field gradient before re-application of the spray.

Debridement Fluids or Sprays:

In some embodiments, the composition described herein can be formulatedas part of a debridement fluid (optionally with suspended particulatesthat are abrasive to a lesion area). In some embodiments, thecomposition described herein can be formulated as part of a debridementspray. As used herein, the term “debridement” generally refers tocomplete or partial removal of a subject's dead, damaged, and/orinfected tissue to improve the healing potential of the remaininghealthy and/or non-infected tissue. By way of example only, a pluralityof engineered microbe-targeting molecules (e.g., microbe-targetingmicroparticles or magnetic microbeads) can be suspended in a debridementfluid or spray, e.g., for use in an orthopedic procedure. Thedebridement fluid or spray containing the engineered microbe-targetingmolecules can be applied to a lesion, an abscess or a wound, where theengineered microbe-targeting microparticles or magnetic microbeads cancapture a microbe (e.g., S. aureus) and/or microbial matter from thelesion, abscess or wound. The debridement fluid or spray can then beremoved from the applied site by vacuum, or suction. In someembodiments, the debridement fluid or spray containing the engineeredmicrobe-targeting magnetic microbeads can be also removed from theapplied site by exposing the applied site to a magnetic field gradient,which can pull or attract the applied microbe-targeting magneticmicrobeads out from the applied site.

Medical Device Coating:

In some embodiments, the composition described herein can be coated on asurface of a medical device, e.g., a fluid delivery device such ashollow fibers, tubing or a spiral mixer in an extracorporeal device, oran implantable device such as an indwelling catheter, chip or scaffold.By way of example only, a plurality of engineered microbe-targetingmolecules can be coated or conjugated to a surface of a fluid deliverydevice such that when a fluid (e.g., blood) flows through the fluiddelivery device coated with engineered microbe-targeting molecules, anymicrobe (e.g., S. aureus) and/or microbial matter present in the fluid(e.g., blood) can be extracted therefrom, thus reducing the chance of amicrobial infection. In another embodiment, a plurality of engineeredmicrobe-targeting molecules coated on a medical device can comprise adetectable label, e.g., a “smart label” described herein, which canprovide a detectable signal when any microbe (e.g., S. aureus) binds toa surface of the medical device, indicating that the medical device hasbeen contaminated and/or infected, and thus is not appropriate for useor implantation.

Provided herein are also methods for removing a microbe and/or microbialmatter from a target area comprising contacting the target area with atleast one composition described herein. As removal of a microbe and/ormicrobial matter from an infected area can treat and/or prevent amicrobial infection or microbial contamination, provided herein alsoinclude methods for treating and/or preventing a microbial infection ormicrobial contamination in a target area. An exemplary method comprisescontacting the target area with a composition. The target area can beanywhere, e.g., an environmental surface or in a body of a subject(e.g., body fluid, and/or tissue). In some embodiments, the methodcomprises contacting the tissue of the subject with any embodiments ofthe composition described herein. In some embodiments, the tissue canhave an open wound, a lesion or an abscess.

In one embodiment, the composition can be formulated for use as a wounddressing described herein.

As the engineered microbe-targeting molecules can localize a microbe(e.g., S. aureus) for easier removal of the microbe from the tissue, insome embodiments, the method can further comprise replacing thepreviously-applied composition in contact with the tissue with a freshcomposition after a period of time. For example, depending on thecondition of the microbial infection and/or specific compositions, thepreviously-applied composition can be replaced every 1 hour, every 2hours, every 3 hours, every 4 hours, every 5 hours, every 6 hours, every8 hours, every 10 hours, every 12 hours, every 16 hours, every 24 hoursor longer.

In some embodiments, the method can further comprise administering anadditional treatment to the tissue. Exemplary additional treatments caninclude, but are not limited to, a negative-pressure treatment, avacuum-assisted debridement, administration of an antimicrobial agent,or any combinations thereof.

Without limitations, the compositions and/or methods of any aspectsdescribed herein can be used to treat and/or prevent a microbialinfection or contamination in vitro, in situ or in vivo. In someembodiments, the compositions and/or methods of any aspects describedherein can be used to treat and/or prevent a microbial infection orcontamination in a fluid or on any surface, including, but not limitedto, a tissue surface, a solid substrate surface, e.g., a medical devicesurface, an environmental surface, or food.

Additionally, in some embodiments where the composition comprises atleast one engineered microbe-targeting molecule conjugated to adetectable label described herein or an imaging agent, can be used toimage an infection in situ, e.g., in a subject or on an environmentalsurface.

S. aureus infections can sometimes be difficult to treat as S. aureushas protein A on its cell surface. Protein A is a wall-anchored proteinwith either four or five domains, each of which can bind to the Fcregion of IgG. The X-ray structure of protein A IgG-binding domains incomplex with the Fc region of IgG has been reported, and residues fromhelix I that are involved in the interaction have been identified andevaluated by site directed mutagenesis. The interaction between proteinA and IgG can coat the surface of the cell with IgG molecules that arein an orientation incorrect to be recognized by the neutrophil Fcreceptor (FIG. 22). This can indicate the anti-phagocytic effect ofprotein A and its role in pathogenesis of S. aureus infections.Protein-A-deficient mutants of S. aureus are reported to be phagocytosedmore efficiently by neutrophils in vitro and show decreased virulence inseveral animal infection models (See, e.g., Fraser T., Nature ReviewsMicrobiology 2005: 3(12):948-58). In accordance with some aspectsprovided herein, the compositions and/or methods described herein can beused to treat or prevent S. aureus microbial infection.

Pharmaceutical Compositions

Some embodiments of the engineered microbe-targeting molecules can beused for therapeutic purposes. For administration to a subject in needthereof, engineered microbe-targeting molecules described herein can beprovided in pharmaceutically acceptable compositions. Accordingly, inyet another aspect, provided herein is a pharmaceutical compositioncomprising at least one engineered microbe-targeting molecule describedherein, and a pharmaceutically acceptable carrier.

Depending on the selected administration route, the compositions orpreparations can be in any form, e.g., a tablet, a lozenge, asuspension, a free-flowing powder, an aerosol, and a capsule. The term“pharmaceutically acceptable,” as used herein, refers to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

As used herein, the term “pharmaceutically acceptable carrier” refers toa pharmaceutically-acceptable material, composition or vehicle foradministration of an active agent described herein. Pharmaceuticallyacceptable carriers include any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like which are compatible with the activity ofthe active agent and are physiologically acceptable to the subject. Someexamples of materials which can serve as pharmaceutically-acceptablecarriers include: (i) sugars, such as lactose, glucose and sucrose; (ii)starches, such as corn starch and potato starch; (iii) cellulose, andits derivatives, such as sodium carboxymethyl cellulose,methylcellulose, ethyl cellulose, microcrystalline cellulose andcellulose acetate; (iv) powdered tragacanth; (v) malt; (vi) gelatin;(vii) lubricating agents, such as magnesium stearate, sodium laurylsulfate and talc; (viii) excipients, such as cocoa butter andsuppository waxes; (ix) oils, such as peanut oil, cottonseed oil,safflower oil, sesame oil, olive oil, corn oil and soybean oil; (x)glycols, such as propylene glycol; (xi) polyols, such as glycerin,sorbitol, mannitol and polyethylene glycol (PEG); (xii) esters, such asethyl oleate and ethyl laurate; (xiii) agar; (xiv) buffering agents,such as magnesium hydroxide and aluminum hydroxide; (xv) alginic acid;(xvi) pyrogen-free water; (xvii) isotonic saline; (xviii) Ringer'ssolution; (xix) ethyl alcohol; (xx) pH buffered solutions; (xxi)polyesters, polycarbonates and/or polyanhydrides; (xxii) bulking agents,such as polypeptides and amino acids (xxiii) serum component, such asserum albumin, HDL and LDL; (xxiv) C2-C12 alcohols, such as ethanol; and(xxv) other non-toxic compatible substances employed in pharmaceuticalformulations. Wetting agents, coloring agents, release agents, coatingagents, sweetening agents, flavoring agents, perfuming agents,preservative and antioxidants can also be present in the formulation.For compositions or preparations described herein to be administeredorally, pharmaceutically acceptable carriers include, but are notlimited to pharmaceutically acceptable excipients such as inertdiluents, disintegrating agents, binding agents, lubricating agents,sweetening agents, flavoring agents, coloring agents and preservatives.Suitable inert diluents include sodium and calcium carbonate, sodium andcalcium phosphate, and lactose, while corn starch and alginic acid aresuitable disintegrating agents. Binding agents may include starch andgelatin, while the lubricating agent, if present, will generally bemagnesium stearate, stearic acid or talc. If desired, the tablets may becoated with a material such as glyceryl monostearate or glyceryldistearate, to delay absorption in the gastrointestinal tract.

Pharmaceutically acceptable carriers can vary in a preparation describedherein, depending on the administration route and formulation. Thecompositions and preparations described herein can be delivered via anyadministration mode known to a skilled practitioner. For example, thecompositions and preparations described herein can be delivered in asystemic manner, via administration routes such as, but not limited to,oral, and parenteral including intravenous, intramuscular,intraperitoneal, intradermal, and subcutaneous. In some embodiments, thecompositions and preparations described herein are in a form that issuitable for injection. In other embodiments, the compositions andpreparations described herein are formulated for oral administration.

When administering parenterally, a composition and preparation describedherein can be generally formulated in a unit dosage injectable form(solution, suspension, emulsion). The compositions and preparationssuitable for injection include sterile aqueous solutions or dispersions.The carrier can be a solvent or dispersing medium containing, forexample, water, cell culture medium, buffers (e.g., phosphate bufferedsaline), polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycol, and the like), suitable mixtures thereof. In someembodiments, the pharmaceutical carrier can be a buffered solution (e.g.PBS).

An oral composition can be prepared in any orally acceptable dosage formincluding, but not limited to, tablets, capsules, emulsions and aqueoussuspensions, dispersions and solutions. Commonly used carriers fortablets include lactose and corn starch. Lubricating agents, such asmagnesium stearate, are also typically added to tablets. For oraladministration in a capsule form, useful diluents include lactose anddried corn starch. When aqueous suspensions or emulsions areadministered orally, the active ingredient can be suspended or dissolvedin an oily phase combined with emulsifying or suspending agents. Ifdesired, certain sweetening, flavoring, or coloring agents can be added.Liquid preparations for oral administration can also be prepared in theform of a dry powder to be reconstituted with a suitable solvent priorto use.

The compositions can also contain auxiliary substances such as wettingor emulsifying agents, pH buffering agents, gelling or viscosityenhancing additives, preservatives, colors, and the like, depending uponthe route of administration and the preparation desired. Standard texts,such as “REMINGTON'S PHARMACEUTICAL SCIENCE”, 17th edition, 1985,incorporated herein by reference, may be consulted to prepare suitablepreparations, without undue experimentation. With respect tocompositions described herein, however, any vehicle, diluent, oradditive used should have to be biocompatible with the active agentsdescribed herein. Those skilled in the art will recognize that thecomponents of the compositions should be selected to be biocompatiblewith respect to the active agent. This will present no problem to thoseskilled in chemical and pharmaceutical principles, or problems can bereadily avoided by reference to standard texts or by simple experiments(not involving undue experimentation).

In some embodiments, the compositions and preparations described hereincan be formulated in an emulsion or a gel. Such gel compositions andpreparations can be implanted locally to a diseased tissue region of asubject.

For in vivo administration, the compositions or preparations describedherein can be administered with a delivery device, e.g., a syringe.Accordingly, an additional aspect described herein provides for deliverydevices comprising at least one chamber with an outlet, wherein the atleast one chamber comprises a pre-determined amount of any compositiondescribed herein and the outlet provides an exit for the compositionenclosed inside the chamber. In some embodiments, a delivery devicedescribed herein can further comprise an actuator to control release ofthe composition through the outlet. Such delivery device can be anydevice to facilitate the administration of any composition describedherein to a subject, e.g., a syringe, a dry powder injector, a nasalspray, a nebulizer, or an implant such as a microchip, e.g., forsustained-release or controlled release of any composition describedherein.

In some embodiments of the products described herein, themicrobe-targeting microparticles described herein itself can be modifiedto control its degradation and thus the release of active agents. Insome embodiments, the engineered microbe-targeting molecules,microbe-targeting microparticles and/or microbe-targeting cellsdescribed herein can be combined with other types of delivery systemsavailable and known to those of ordinary skill in the art. They include,for example, polymer-based systems such as polylactic and/orpolyglycolic acids, polyanhydrides, polycaprolactones, copolyoxalates,polyesteramides, polyorthoesters, polyhydroxybutyric acid, and/orcombinations thereof. Microcapsules of the foregoing polymers containingdrugs are described in, for example, U.S. Pat. No. 5,075,109. Otherexamples include nonpolymer systems that are lipid-based includingsterols such as cholesterol, cholesterol esters, and fatty acids orneukal fats such as mono-, di- and triglycerides; hydrogel releasesystems; liposome-based systems; phospholipid based-systems; silasticsystems; peptide based systems; or partially fused implants. Specificexamples include, but are not limited to, erosional systems in which thecomposition is contained in a form within a matrix (for example, asdescribed in U.S. Pat. Nos. 4,452,775, 4,675,189, 5,736,152, 4,667,014,4,748,034 and −29 5,239,660), or diffusional systems in which an activecomponent controls the release rate (for example, as described in U.S.Pat. Nos. 3,832,253, 3,854, 480, 5,133,974 and 5,407,686). Theformulation may be as, for example, microspheres, hydrogels, polymericreservoirs, cholesterol matrices, or polymeric systems. In someembodiments, the system may allow sustained or controlled release of thecomposition to occur, for example, through control of the diffusion orerosion/degradation rate of the formulation containing the composition.In addition, a pump-based hardware delivery system can be used todeliver one or more embodiments of the compositions or preparationsdescribed herein. Use of a long-term sustained release formulations orimplants can be particularly suitable for treatment of some infections.Long-term release, as used herein, means that a formulation or animplant is made and arranged to deliver compositions or preparationsdescribed herein at a therapeutic level for at least 30 days, or atleast 60 days. In some embodiments, the long-term release refers to aformulation or an implant being configured to deliver an active agent ata therapeutic level over several months.

Regeneration of Microbe-Binding Substrates (e.g., Microbe-BindingMicrobeads)

In some applications, an artisan may want to detach or release apathogen captured by or bound to an engineered microbe-targetingmolecule. As discussed herein, calcium ions are involved in bindinginteractions of the engineered microbe-targeting molecules describedherein with microbe surface. A skilled artisan will appreciate thatdetaching the pathogen from support bound microbe-targeting moleculealso regenerates the support bound microbe-targeting molecule.

Accordingly, disclosed herein are methods for inhibiting Ca²⁺ assistedinteractions between two components, e.g., in a complex, by reducing theamount of Ca²⁺ ions available for the interactions. This can beaccomplished by contacting or incubating the complex with a buffer orsolution comprising a chelating agent which chelates calcium ions.Exemplary chelating agents include, but are not limited to,1,2-Bis(2-Aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid;Ethylenediaminetetraacetic acid (EDTA); Ethyleneglycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid; and Ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid.

For some uses, chelating agents can be problematic. For example,chelating agents such as EDTA and EGTA can be harsh or dangerous tobiological samples. Accordingly, the inventors have also discoveredalternative methods for reducing the amount of Ca²⁺ ions available forassisting in complex formation. In one example, the complex can becontacted or incubated with a low pH buffer. Without wishing to be boundby a theory, low pH buffer protonates the negatively charged carboxylgroups (glutamate side chains) on the engineered microbe-targetingmolecules that are responsible for binding calcium. Protonating theseside chains can remove their negative charge, can remove their abilityto bind to positively charged calcium ions. In some embodiments, the lowpH buffer is of about pH 6.75, about pH 6.5, about pH 6.25, about pH 6,about pH 5.75, about pH 5.5, about pH 5.25, about pH 5, about pH 4.5,about pH 4, about pH 3.5, about pH 3, about pH 2.5 or lower. In oneembodiment, buffer is of pH about 2.8. In some embodiments, the low pHbuffer can further comprise a chelating agent.

Alternatively or in addition to a low pH buffer, one can also use abuffer in which calcium is not soluble. For example, calcium caninteract with one or more components of the buffer and can precipitateout of the buffer solution. Thus, contacting or incubating the complexin such a buffer can lead to precipitation of the calcium ions makingthem unavailable for the necessary interaction with the targetingmolecule-microbe interface. Generally, buffers in which calcium is notsoluble include an anion which forms a salt with the Ca²⁺ ion. Thusformed salt is less soluble in the solvent of the buffer. Exemplaryanion which produce insoluble salts with Ca²⁺ include, but are notlimited to, phosphates, oxalates, carbonates, sulfates, fluorides,gluconic acid, oxido-trioxo-manganese, stearic acid, and the like. Insome embodiments, the buffer can further comprise a chelating agent.

In some embodiments, the buffer is a 0.2M glycine buffer of pH 2.8. Insome embodiments, the buffer is a 0.1M sodium phosphate buffer of pH6.0.

Many of the calcium salts become more insoluble at elevated temperature.Accordingly, during detachment of the pathogen, temperature of thebuffer can be increased or decrease. In some embodiments, the buffer isheated during detachment of the pathogen. In some other embodiments, thebuffer is cooled during detachment of the pathogen. Temperature of thebuffer can be increased or decreased by at least 5° C., at least 10° C.,at least 15° C., at least 20° C., at least 25° C. or more relative toroom temperature.

The method described herein for inhibiting Ca²⁺ assisted interactionsbetween two components can also be used for detaching a pathogen from anengineered microbe-targeting molecule. For example, thepathogen-targeting molecule complex can be contacted or incubated with alow pH buffer or with a buffer in which calcium is not soluble.

In one embodiment, a bound pathogen can be detached from a targetingmolecule using a 0.2M glycine buffer at pH 2.8. In another embodiment, abound pathogen can be detached from a targeting molecule using a 0.1Msodium phosphate buffer at pH 6.0.

If the targeting molecule with the bound pathogen is attached to asupport surface, e.g., a microparticle or a magnetic microparticle, thepathogen can be detached by incubating or contacting the support with alow pH buffer or a buffer in which calcium is not soluble. Thus providedherein is also a method for detaching a microbe from a support boundmicrobe-targeting molecule. The method comprising contacting, washing,or incubating the support bound pathogen with a low pH buffer or abuffer in which calcium in insoluble. After a predetermined time (e.g.,5 mins, 10 mins, 15 mins, 30 mins, 25 mins, 30 mins, 35 mins, 40 mins,45 mins, 50 mins, 55 mins, 1 hour, 1.25 hours, 1.5 hours, 2 hours, 3hours, 4 hours, 5 hours, 6 hours or more) has passed, the buffer can beremoved and the support optionally washed one or more times. Withoutwishing to be bound by a theory, this regenerates the support boundtargeting molecules for binding with pathogens in sample. In otherwords, detaching the microbes allows one to re-use the support boundtargeting molecules. The detached pathogens can be used for analysis,detection or for any other use.

Kits

Kits for capturing, detecting and/or determining the presence or absenceof a microbe and/or microbial matter in a sample are also providedherein. In some embodiments, the kit can comprise: (a) one or morecontainers containing a population of engineered microbe-targetingmolecules described herein; and (b) at least one reagent. In theseembodiments, a user can generate their own microbe-targeting substratesby conjugating the provided engineered microbe-targeting molecules totheir desired substrate, e.g., using any art-recognized conjugationchemistry and/or methods described herein. In such embodiments, thereagent can include, but is not limited to, a coupling agent forconjugation of engineered microbe-targeting molecules to a substrate. Insome embodiments, the kit can further comprise one or more substrates(e.g., microbeads such as magnetic microbeads) to which the engineeredmicrobe-targeting molecules described herein are conjugated. In suchembodiments, a user can further modify the surface chemistry of theprovided substrate prior to conjugation of the engineeredmicrobe-targeting molecules to the substrate.

In other embodiments, the kit can provide microbe-targeting substratesthat are ready for use. Accordingly, in these embodiments, the kit cancomprise: (a) one or more microbe-targeting substrates described herein;and (b) at least one reagent. In some embodiments, the microbe-targetingsubstrate can include one or more microbe-binding dipsticks, e.g., asdescribed herein. In other embodiments, the microbe-targeting substratecan include a population of microbe-targeting microbeads (including, butnot limited to, polymeric microbeads and magnetic microbeads). In someembodiments, the microbe-targeting substrate can include a population ofmicrobe-targeting magnetic microbeads. The microbe-targeting microbeadsor microbe-targeting magnetic microbeads can be provided in one or moreseparate containers, if desired. In some embodiments, the population ofthe microbe-targeting microbeads or magnetic microbeads contained in oneor more containers can be lyophilized.

In some embodiments of any aspects of the kits described herein, thepopulation of the microbeads or microbe-targeting microbeads cancomprise at least one distinct subset of the microbeads ormicrobe-targeting microbeads, respectively. For example, each distinctsubset of the microbeads or microbe-targeting microbeads can be providedin a separate container. In some embodiments, the distinct subset of themicrobeads or microbe-targeting microbeads can have a size. In someembodiments, the distinct subset of microbe-targeting microbeads cancomprise on their surfaces a different density of engineeredmicrobe-targeting molecules from the rest of the population. In theseembodiments, two or more subsets of the microbe-targeting microbeshaving different sizes and/or different coating density of theengineered microbe-binding molecules can be used to detect anddifferentiate microbes of different classes and/or sizes, e.g.,employing the methods described herein. In some embodiments, thedistinct subset of microbe-targeting substrates, e.g., microbe-targetingmicrobeads, can comprise a different carbohydrate recognition domainfrom the others.

In some embodiments of any aspects of the kits described herein, thesubstrates (e.g., microbeads) or microbe-targeting substrates (e.g.,microbe-targeting microbeads) can further comprise a detection label. Byway of example only, depending on the choice of detection methods, eachdistinct subset of the microbeads can comprise a unique detection labelor the same detection label. For example, if each distinct subset of themicrobe-targeting microbeads is used in a different sampling well, thesame detection label can be used on the microbe-targeting microbeads.However, if it is desirable to detect multiple differentmicrobe-targeting microbeads in the same well, it is preferably to haveeach distinct subset of microbe-targeting microbeads comprising adistinct detection label.

Detectable labels suitable for use in any kits provided herein includeany composition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Anyart-recognized detectable labels or the ones described herein can beincluded in the kits described herein.

Means of detecting such labels are well known to those of skill in theart and exemplary detection methods are described herein. For example,radiolabels can be detected using photographic film or scintillationcounters, fluorescent markers can be detected using a photo-detector todetect emitted light. Enzymatic labels are typically detected byproviding the enzyme with an enzyme substrate and detecting the reactionproduct produced by the action of the enzyme on the enzyme substrate,and calorimetric labels can be detected by visualizing the coloredlabel.

In some embodiments of any aspects described herein, the kits canfurther comprise one or more containers containing a population ofdetectable labels, wherein the detectable label is conjugated to amolecule. In some embodiments, at least one of the containers cancontain a distinct population of detectable labels.

The molecule conjugated to a detectable label can be any molecule thatbinds to a microbe of interest. For example, in some embodiments, themolecule conjugated to a detectable label can comprise the samecarbohydrate recognition domains as used in the microbe-targetingsubstrates (e.g., microbe-targeting magnetic microbeads). In suchembodiments, at least one population of the molecule-detectable labelconjugate can comprise at least one carbohydrate recognition domain or afragment thereof, e.g., derived from mannose-binding lectin or at leasta portion of the CRD domain, e.g., encoded by SEQ ID NO. 4, or afragment thereof. In some embodiments, the molecule conjugated to adetectable label can further comprise a Fc region of an immunoglobulin.In alternative embodiments, the molecule conjugated to a detectablelabel can comprise an antibody specific to at least one genus, species,or type/class of microbes (e.g., gram-positive vs. gram-negativemicrobes; protein A-expressing or protein G-expressing microbes vs.protein A- or protein G-negative microbes) recognized by themicrobe-targeting molecules described herein, or an antibody specific toat least one type of carbohydrate recognition domain (e.g., C-typelectins vs. S-type lectins) employed in the microbe-targeting moleculesdescribed herein. However, the antibody can also be a common antibodythat binds to all the microbes or pathogens recognized by themicrobe-targeting molecules provided in the kit. Without limitations, amolecule attached to a detectable label can also include any ligandtargeting microbial cell surface proteins or receptors, includingcarbohydrates, lipids, lectins, aptamers, protein, peptides, nucleicacid, polynucleotides, antibody or a portion thereof, an antibody-likemolecule, peptidomimetic, and any combinations thereof.

In some embodiments, at least one of the containers can contain adistinct population of the molecule-detectable label conjugate asdescribed earlier. The distinct population of the molecule-detectablelabel conjugate can contain a unique molecule with the detectable labelsame as others, or a conjugate comprising a distinct detectable label(e.g., a unique fluorescent molecule) and a distinct molecule. As eachdistinct detectable label can identify the associated protein,conjugates comprising a distinct detectable label associated with adistinct molecule can allow detecting in a single sample at least two ormore distinct populations of the engineered microbe-targeting substrates(e.g., microbe-targeting magnetic microbeads); for example, eachdistinct population of the engineered microbe-targeting magneticmicrobeads can bind to a distinct genus or species or type/size of amicrobe. In alternative embodiments, the molecule-detectable labelconjugates in each of the containers can comprise the same detectablelabel. For example, the detectable label can comprise an enzyme (e.g.,horseradish peroxidase or alkaline phosphatase) that produces a colorchange in the presence of an enzyme substrate. In such embodiments, thekit can further comprise one or more containers containing an enzymesubstrate that changes color in the presence of the enzyme.

In one embodiment, the microbe-targeting substrate provided in the kitcan include a dipstick or test strip or membrane containing one or moreengineered microbe-targeting molecules, e.g., microbe-binding dipstickor membrane described herein. In this embodiment, the kit can comprise1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, 100, 150, 200 or moremicrobe-binding dipsticks or test strips described herein. These kitscomprising the microbe-binding dipsticks or test strips can be used as adiagnostic or probe for a microbe anywhere, e.g., at home, in clinics orhospitals, on emergency vehicles, in outdoor environments, in foodprocessing plants, and anywhere in need of microbe capture and/ordetection.

In some embodiments, each microbe-targeting substrate or productdescribed herein, e.g., each microbe-binding dipstick or membrane, canbe individually packaged to maintain their sterility. In someembodiments, two or more products (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 25, 50, or more products such as microbe-binding dipsticks ormembranes) can be packaged into one single unit. In such embodiments,users can sterilize any unused products after opening, e.g., with UVradiation, high temperature, gamma-radiation, ethylene oxidesterilization or any other known methods that would not significantlyaffect the activity of the engineered microbe-targeting molecules formicrobe detection.

In other embodiments, the microbe-targeting substrate provided in thekit can include a population of microbe-targeting microbeads or magneticmicrobeads. In some embodiments, the microbe-targeting microbeads ormagnetic microbeads can be lyophilized.

Depending on the configuration/combination of the molecule-detectablelabel conjugates provided in the kit, different populations of themicrobe-targeting microbeads or magnetic microbeads can be mixedtogether with a test sample in a single reaction, or differentpopulations each can be applied separately to different aliquots of thesame test sample. After contacting the test sample with themicrobe-targeting microbeads or magnetic microbeads, any microbes orpathogens recognized by the microbe-targeting molecules will bind to themicrobe-targeting microbeads or magnetic microbeads.

In some embodiments, the kit can further comprise at least one bloodcollection container or any equivalent sample container or chamber,including at least 1, at least 2, at least 3, at least 4, at least 5, atleast 6, at least 7, at least 8, at least 9, at least 10, at least 15,at least 20 blood collection containers or equivalent sample containersor chambers. In some embodiments, the population of themicrobe-targeting microbeads or magnetic microbeads can be pre-loaded inat least one blood collection container. In some embodiments, the bloodcollection container can further comprise an anti-coagulant agentdescribed herein. In some embodiments, a blood sample can be directlyadded to such blood collection container containing a population of themicrobe-targeting and/or microbe-binding microbeads or magneticmicrobeads for carrying out a microbe detection assay, e.g., asdescribed in Example 10 and FIG. 14. While Example 10 and FIG. 14illustrates the use of microbe-targeting magnetic microbeads for captureof microbes, an ordinary artisan will readily appreciate that someembodiments of the microbe-targeting microbeads (without magneticproperties) described herein can also be applicable for the assay. Forexample, instead of using a magnet to collect the microbe-targetingmagnetic microbeads after contact with a test sample (e.g., a bloodsample), the microbe-targeting microbeads (without magnetic properties)can also be collected, e.g., by filtration, centrifugation or any othermethods known in the art.

In some embodiments where the kits comprise microbe-targeting magneticmicrobeads, the kits can further comprise a magnet adapted for use withthe assay for isolation of the microbe-targeting magnetic microbeadsfrom a test sample. For example, if the assay is carried out in a bloodcollection tube, the magnet can be adapted for use with the bloodcollection tube, e.g., a magnet can be designed to be a magnet collarsurrounding the blood collection tube to immobilize or isolate themicrobe-targeting magnetic microbeads from a test sample or an assaybuffer.

In any aspects of the kits provided herein, the kits can furthercomprise a portable readout machine or device, e.g., to determine anddisplay the signal produced from the assay performed with the kit. Forexample, the readout machine or device can detect a colorimetric signaland/or a fluorescent signal produced from the assay of pathogendetection performed with the kits described herein.

In any aspects of the kits described herein, the kits can furtherinclude a reference for comparison with a readout determined from a testsample. An exemplary reference can be a strip or a chart showingdifferent colors corresponding to various extents or degrees of amicrobial infection.

Depending on different embodiments of the engineered microbe-targetingmolecules and/or products provided in the kits, some embodiments of anyaspects of the kits described herein can further comprise an additionalagent. For example, in some embodiments where the engineeredmicrobe-targeting molecules present on the substrate are unlabeled, thekit can further comprise one or more containers containing a populationof detectable labels described earlier, each of which is conjugated to atargeting agent specific for a microbe, e.g., without limitations, oneor more embodiments of an engineered microbe-targeting molecule or afragment thereof, an antibody specific for at least one microbe (e.g.,antibodies specific for Gram-positive microbes such as anti-LTAantibodies, antibodies specific for Gram-negative microbes such asanti-LPS antibodies, or antibodies specific for fungus, and anycombinations thereof). The use of an additional targeting agent specificfor a microbe conjugated to a detectable label can not only facilitatethe detection of microbes or pathogens, but can also increase thespecificity of the detection for a microbe or a pathogen.

In any aspects of the kits provided herein, when the detection labelincludes an enzyme (e.g., horseradish peroxidase, alkaline phosphataseand any others commonly used for colorimetric detection), the kits canfurther comprise one or more containers containing an enzyme substratethat produces a color change in the presence of the enzyme. One of skillin the art can readily recognize an appropriate enzyme substrate for anyart-recognized enzymes used for colorimetric detection. By way ofexample only, an exemplary substrate for alkaline phosphatase caninclude BCIP/NBT or PNPP (p-Nitrophenyl Phosphate, Disodium Salt); anexemplary substrate for horseradish peroxidase can include TMB.

In any aspects of the kits provided herein, the at least one reagent canbe a wash buffer, a dilution buffer, a stop buffer, e.g., to stop thecolor development, a buffer solution containing a chelating agentdescribed herein, or any combinations thereof. In one embodiment, atleast one of the reagents provided in the kit can include at least onebuffered solution containing a chelating agent. The chelating agent canbe used to chelate any ions (e.g., divalent ions) present in the testsamples or assay buffer, e.g., for inhibiting calcium-dependent bindingof certain microbes, but not others, to some embodiments of themicrobe-binding molecules described herein. Accordingly, such kit can beused to distinguish one microbe (e.g., S. aureus) from another (e.g., E.coli) in a test sample, e.g. employing some embodiments of the methoddescribed herein.

In any aspects of the kits provided herein, the kits can furthercomprise at least one microtiter plate, e.g., for performing thereaction and the detection.

In addition to the above mentioned components, any embodiments of thekits described herein can include informational material. Theinformational material can be descriptive, instructional, marketing orother material that relates to the methods described herein and/or theuse of the aggregates for the methods described herein. For example, theinformational material can describe methods for using the kits providedherein to perform an assay for pathogen or microbe capture and/ordetection. The kit can also include an empty container and/or a deliverydevice, e.g., which can be used to deliver a test sample to a testcontainer.

The informational material of the kits is not limited in its form. Inmany cases, the informational material, e.g., instructions, is providedin printed matter, e.g., a printed text, drawing, and/or photograph,e.g., a label or printed sheet. However, the informational material canalso be provided in other formats, such as Braille, computer readablematerial, video recording, or audio recording. In another embodiment,the informational material of the kit is a link or contact information,e.g., a physical address, email address, hyperlink, website, ortelephone number, where a user of the kit can obtain substantiveinformation about the formulation and/or its use in the methodsdescribed herein. Of course, the informational material can also beprovided in any combination of formats.

In some embodiments, the kit can contain separate containers, dividersor compartments for each component and informational material. Forexample, each different component can be contained in a bottle, vial, orsyringe, and the informational material can be contained in a plasticsleeve or packet. In other embodiments, the separate elements of the kitare contained within a single, undivided container. For example, acollection of the magnetic microbeads is contained in a bottle, vial orsyringe that has attached thereto the informational material in the formof a label.

In general, the kits described herein can be used to separate, remove,and/or detect a microbe present in a test sample. In some embodiments,the kits can be used to differentiate between different microbe species,classes, and/or sizes, by employing the methods and/or assays describedherein. By way of example only, some embodiments of the kits can be usedto detect the presence or absence of any protein A-expressing microbe orany protein G-expressing microbe in a test sample. Accordingly, someembodiments of the kits described herein can be used to detect ordetermine the presence or absence of at least one staphylococcusspecies, excluding S. epidermidis, in a test sample. In one embodiment,the assays, methods, and kits described herein can be used to detect ordetermine the presence or absence of S. aureus in a test sample. In someembodiments, the assays, methods, and kits described herein can be usedto detect or determine the presence or absence of at least onestreptococci species in a test sample.

In some embodiments, the kits described herein can be used to screen apharmaceutical product (e.g., a drug, a therapeutic agent, or an imagingagent), and/or a medical device (including, but not limited to,implantable devices) for the presence or absence of microbial matter(including, but not limited to, endotoxins secreted by a microbe).

Test Sample

In accordance with various embodiments described herein, a test sampleor sample, including any fluid or specimen (processed or unprocessed),that is suspected of comprising a microbe and/or microbial matter can besubjected to an assay or method, kit and system described herein. Thetest sample or fluid can be liquid, supercritical fluid, solutions,suspensions, gases, gels, slurries, and combinations thereof. The testsample or fluid can be aqueous or non-aqueous.

In some embodiments, the test sample can be an aqueous fluid. As usedherein, the term “aqueous fluid” refers to any flowable water-containingmaterial that is suspected of comprising a microbe and/or microbialmatter.

In some embodiments, the test sample can include a biological fluidobtained from a subject. Exemplary biological fluids obtained from asubject can include, but are not limited to, blood (including wholeblood, plasma, cord blood and serum), lactation products (e.g., milk),amniotic fluids, sputum, saliva, urine, semen, cerebrospinal fluid,bronchial aspirate, perspiration, mucus, liquefied feces, synovialfluid, lymphatic fluid, tears, tracheal aspirate, and fractions thereof.In some embodiments, a biological fluid can include a homogenate of atissue specimen (e.g., biopsy) from a subject.

In some embodiments, the biological fluid sample obtained from asubject, e.g., a mammalian subject such as a human subject or a domesticpet such as a cat or dog, can contain cells from the subject. In otherembodiments, the biological fluid sample can contain non-cellularbiological material, such as non-cellular fractions of blood, saliva, orurine, which can be used to measure plasma/serum biomarker expressionlevels.

The biological fluid sample can be freshly collected from a subject or apreviously collected sample. In some embodiments, the biological fluidsample used in the assays and/or methods described herein can becollected from a subject no more than 24 hours, no more than 12 hours,no more than 6 hours, no more than 3 hours, no more than 2 hours, nomore than 1 hour, no more than 30 mins or shorter.

In some embodiments, the biological fluid sample or any fluid sampledescribed herein can be treated with a chemical and/or biologicalreagent described herein prior to use with the assays and/or methodsdescribed herein. In some embodiments, at least one of the chemicaland/or biological reagents can be present in the sample container beforea fluid sample is added to the sample container. For example, blood canbe collected into a blood collection tube such as VACUTAINER®, which hasalready contained heparin. Examples of the chemical and/or biologicalreagents can include, without limitations, surfactants and detergents,salts, cell lysing reagents, anticoagulants, degradative enzymes (e.g.,proteases, lipases, nucleases, collagenases, cellulases, amylases), andsolvents such as buffer solutions.

In some embodiments, the test sample can include a fluid or specimenobtained from an environmental source, e.g., but not limited to, watersupplies (including wastewater), ponds, rivers, reservoirs, swimmingpools, soils, food processing and/or packaging plants, agriculturalplaces, hydrocultures (including hydroponic food farms), pharmaceuticalmanufacturing plants, animal colony facilities, and any combinationsthereof.

In some embodiments, the test sample can include a fluid (e.g., culturemedium) from a biological culture. Examples of a fluid (e.g., culturemedium) obtained from a biological culture includes the one obtainedfrom culturing or fermentation, for example, of single- or multi-cellorganisms, including prokaryotes (e.g., bacteria) and eukaryotes (e.g.,animal cells, plant cells, yeasts, fungi), and including fractionsthereof. In some embodiments, the test sample can include a fluid from ablood culture. In some embodiments, the culture medium can be obtainedfrom any source, e.g., without limitations, research laboratories,pharmaceutical manufacturing plants, hydrocultures (e.g., hydroponicfood farms), diagnostic testing facilities, clinical settings, and anycombinations thereof.

In some embodiments, the test sample can include a media or reagentsolution used in a laboratory or clinical setting, such as forbiomedical and molecular biology applications. As used herein, the term“media” refers to a medium for maintaining a tissue, an organism, or acell population, or refers to a medium for culturing a tissue, anorganism, or a cell population, which contains nutrients that maintainviability of the tissue, organism, or cell population, and supportproliferation and growth.

As used herein, the term “reagent” refers to any solution used in alaboratory or clinical setting for biomedical and molecular biologyapplications. Reagents include, but are not limited to, salinesolutions, PBS solutions, buffered solutions, such as phosphate buffers,EDTA, Tris solutions, and any combinations thereof. Reagent solutionscan be used to create other reagent solutions. For example, Trissolutions and EDTA solutions are combined in specific ratios to create“TE” reagents for use in molecular biology applications.

In some embodiments, the test sample can be a non-biological fluid. Asused herein, the term “non-biological fluid” refers to any fluid that isnot a biological fluid as the term is defined herein. Exemplarynon-biological fluids include, but are not limited to, water, saltwater, brine, buffered solutions, saline solutions, sugar solutions,carbohydrate solutions, lipid solutions, nucleic acid solutions,hydrocarbons (e.g. liquid hydrocarbons), acids, gasoline, petroleum,liquefied samples (e.g., liquefied samples), and mixtures thereof.

Exemplary Microbes or Pathogens

As used herein, the term “microbes” or “microbe” generally refers tomicroorganism(s), including bacteria, fungi, protozoan, archaea,protists, e.g., algae, and a combination thereof. The term “microbes”encompasses both live and dead microbes. The term “microbes” alsoincludes pathogenic microbes or pathogens, e.g., bacteria causingdiseases such as plague, tuberculosis and anthrax; protozoa causingdiseases such as malaria, sleeping sickness and toxoplasmosis; fungicausing diseases such as ringworm, candidiasis or histoplasmosis; andbacteria causing diseases such as sepsis.

Microbe-Induced Diseases:

In some other embodiments, the engineered microbe-targeting molecules orsubstrates, products and kits described herein can be used to detect orbind to the following microbes that causes diseases and/or associatedmicrobial matter: Bartonella henselae, Borrelia burgdorferi,Campylobacter jejuni, Campylobacterfetus, Chlamydia trachomatis,Chlamydia pneumoniae, Chylamydia psittaci, Simkania negevensis,Escherichia coli (e.g., 0157:H7 and K88), Ehrlichia chafeensis,Clostridium botulinum, Clostridium perfringens, Clostridium tetani,Enterococcus faecalis, Haemophilius influenzae, Haemophilius ducreyi,Coccidioides immitis, Bordetella pertussis, Coxiella burnetii,Ureaplasma urealyticum, Mycoplasma genitalium, Trichomatis vaginalis,Helicobacter pylori, Helicobacter hepaticus, Legionella pneumophila,Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacteriumafricanum, Mycobacterium leprae, Mycobacterium asiaticum, Mycobacteriumavium, Mycobacterium celatum, Mycobacterium celonae, Mycobacteriumfortuitum, Mycobacterium genavense, Mycobacterium haemophilum,Mycobacterium intracellulare, Mycobacterium kansasii, Mycobacteriummalmoense, Mycobacterium marinum, Mycobacterium scrofulaceum,Mycobacterium simiae, Mycobacterium szulgai, Mycobacterium ulcerans,Mycobacterium xenopi, Corynebacterium diptheriae, Rhodococcus equi,Rickettsia aeschlimannii, Rickettsia africae, Rickettsia conorii,Arcanobacterium haemolyticum, Bacillus anthracis, Bacillus cereus,Lysteria monocytogenes, Yersinia pestis, Yersinia enterocolitica,Shigella dysenteriae, Neisseria meningitides, Neisseria gonorrhoeae,Streptococcus bovis, Streptococcus hemolyticus, Streptococcus mutans,Streptococcus pyogenes, Streptococcus pneumoniae, Staphylococcus aureus,Staphylococcus epidermidis, Staphylococcus pneumoniae, Staphylococcussaprophyticus, Vibrio cholerae, Vibrio parahaemolyticus, Salmonellatyphi, Salmonella paratyphi, Salmonella enteritidis, Treponema pallidum,Human rhinovirus, Human coronavirus, Dengue virus, Filoviruses (e.g.,Marburg and Ebola viruses), Hantavirus, Rift Valley virus, Hepatitis B,C, and E, Human Immunodeficiency Virus (e.g., HIV-1, HIV-2), HHV-8,Human papillomavirus, Herpes virus (e.g., HV-I and HV-II), Human T-celllymphotrophic viruses (e.g., HTLV-I and HTLV-II), Bovine leukemia virus,Influenza virus, Guanarito virus, Lassa virus, Measles virus, Rubellavirus, Mumps virus, Chickenpox (Varicella virus), Monkey pox, EpsteinBahr virus, Norwalk (and Norwalk-like) viruses, Rotavirus, ParvovirusB19, Hantaan virus, Sin Nombre virus, Venezuelan equine encephalitis,Sabia virus, West Nile virus, Yellow Fever virus, causative agents oftransmissible spongiform encephalopathies, Creutzfeldt-Jakob diseaseagent, variant Creutzfeldt-Jakob disease agent, Candida, Cryptcooccus,Cryptosporidium, Giardia lamblia, Microsporidia, Plasmodium vivax,Pneumocystis carinii, Toxoplasma gondii, Trichophyton mentagrophytes,Enterocytozoon bieneusi, Cyclospora cayetanensis, Encephalitozoonhellem, Encephalitozoon cuniculi, among other viruses, bacteria,archaea, protozoa, and fungi).

In some embodiments, the engineered microbe-targeting molecules orsubstrates, products and kits described herein can be used todifferentiate a protein A-expressing or protein G-expressing microbefrom protein A- and protein G-negative microbes (e.g., E. coli) byemploying the methods or assays described herein.

In some embodiments, a protein A-expressing microbe includesStaphylococcus species. Examples of Staphylococcus species include, butare not limited to, S. aureus group (e.g., S. aureus, S. simiae), S.auricularis group (e.g., S. auricularis), S. carnosus group (e.g., S.carnosus, S. condimenti, S. massiliensis, S. piscifermentans, S.simulans), S. epidermidis group (e.g., S. capitis, S. caprae, S.epidermidis, S. saccharolyticus), S. haemolyticus group (e.g., S.devriesei, S. haemolyticus, S. hominis), S. hyicus-intermedius group(e.g., S. chromogenes, S. fells, S. delphini, S. hyicus, S. intermedius,S. lutrae, S. microti, S. muscae, S. pseudintermedius, S. rostri, S.schleiferi), S. lugdunensis group (e.g., S. lugdunensis), S.saprophyticus group (e.g., S. arlettae, S. cohnii, S. equorum, S.gallinarum, S. kloosii, S. leei, S. nepalensis, S. saprophyticus, S.succinus, S. xylosus), S. sciuri group (e.g., S. fleurettii, S. lentus,S. sciuri, S. stepanovicii, S. vitulinus), S. simulans group (e.g., S.simulans), and S. warneri group (e.g., S. pasteuri, S. warneri).

In some embodiments, S. aureus can be differentiated from a protein A-and protein G-negative microbe (e.g., E. coli) using the assays and/ormethods described herein.

In some embodiments, S. aureus can be differentiated from S. epidermidisusing the assays and/or methods described herein.

In some embodiments, S. epidermidis cannot be differentiated from aprotein A- and protein G-negative microbe (e.g., E. coli) using theassays and/or methods described herein.

In some embodiments, a protein G-expressing microbe includesStreptococcus species. Examples of Streptococcus species can include,but are not limited to, alpha-hemolytic including Pneumococci (e.g., S.pneumonia), and the Viridans group (e.g., S. mutans, S. mitis, S.sanguinis, S. salivarius, S. salivarius ssp. thermophilus, S.constellatus); and beta-hemolytic including Group A (e.g., S. pyogenes),Group B (e.g., S. agalactiae), Group C (e.g., S. equi, and S.zooepidemicus), Group D (e.g., enterococci, Streptococcus bovis andStreptococcus equinus), Group F streptococci, and Group G streptococci.

In some embodiments, a protein G-expressing microbe includes Group C andGroup G streptococci.

One skilled in the art can understand that the engineeredmicrobe-targeting molecules or substrates, products and kits describedherein can be used to target any microorganism with a microbesurface-binding domain described herein modified for each microorganismof interest. A skilled artisan can determine the cell-surface proteinsor carbohydrates for each microorganism of interest using anymicrobiology techniques known in the art.

Biofilm:

Accordingly, in some embodiments, the microbe-targeting molecules orsubstrates, products and kits herein can be used to detect microbesand/or associated microbial matter present in a biofilm or to treatequipment surfaces to prevent or inhibit formation of a biofilm. Forexample, Listeria monocytogenes can form biofilms on a variety ofmaterials used in food processing equipment and other food and non-foodcontact surfaces (Blackman, J Food Prot 1996; 59:827-31; Frank, J FoodProt 1990; 53:550-4; Krysinski, J Food Prot 1992; 55:246-51; Ronner, JFood Prot 1993; 56:750-8). Biofilms can be broadly defined as microbialcells attached to a surface, and which are embedded in a matrix ofextracellular polymeric substances produced by the microorganisms.Biofilms are known to occur in many environments and frequently lead toa wide diversity of undesirable effects. For example, biofilms causefouling of industrial equipment such as heat exchangers, pipelines, andship hulls, resulting in reduced heat transfer, energy loss, increasedfluid frictional resistance, and accelerated corrosion. Biofilmaccumulation on teeth and gums, urinary and intestinal tracts, andimplanted medical devices such as catheters and prostheses frequentlylead to infections (Characklis W G. Biofilm processes. In: Characklis WG and Marshall K C eds. New York: John Wiley & Sons, 1990:195-231;Costerton et al., Annu Rev Microbiol 1995; 49:711-45). In someembodiments, the engineered microbe-targeting microparticles, e.g.,encapsulating a drug or a chemical for treatment of a biofilm, can besprayed on contaminated equipment surfaces. The bacteria present in thebiofilm bind to the microbe-targeting microparticles, which release thedrug to treat the bacteria for targeted drug delivery.

In addition, L. monocytogenes attached to surfaces such as stainlesssteel and rubber, materials commonly used in food processingenvironments, can survive for prolonged periods (Helke and Wong, J FoodProt 1994; 57:963-8). This would partially explain their ability topersist in the processing plant. Common sources of L. monocytogenes inprocessing facilities include equipment, conveyors, product contactsurfaces, hand tools, cleaning utensils, floors, drains, walls, andcondensate (Tomkin et al., Dairy, Food Environ Sanit 1999; 19:551-62;Welbourn and Williams, Dairy, Food Environ Sanit 1999; 19:399-401). Insome embodiments, the engineered microbe-targeting molecules can beconfigured to include a “smart label”, which is undetectable whenconjugated to the engineered microbe-targeting molecules, but produces acolor change when released from the engineered molecules in the presenceof a microbe enzyme. Thus, when a microbe binds to the engineeredmicrobe-targeting molecules, the microbe releases enzymes that releasethe detectable label from the engineered molecules. An observation of acolor change indicates a risk for bacteria contamination on a particularsurface, and thus some embodiments of the engineered microbe-targetingmolecules and products can be used for early detection of biofilmformation.

Plant Microbes:

In still further embodiments, the engineered microbe-targeting moleculesor substrates and products described herein can be used to target plantmicrobes and/or associated microbial matter. Plant fungi have causedmajor epidemics with huge societal impacts. Examples of plant fungiinclude, but are not limited to, Phytophthora infestans, Crinipellisperniciosa, frosty pod (Moniliophthora roreri), oomycete Phytophthoracapsici, Mycosphaerella fijiensis, Fusarium Ganoderma spp fungi andPhytophthora. An exemplary plant bacterium includes Burkholderiacepacia. Exemplary plant viruses include, but are not limited to,soybean mosaic virus, bean pod mottle virus, tobacco ring spot virus,barley yellow dwarf virus, wheat spindle streak virus, soil born mosaicvirus, wheat streak virus in maize, maize dwarf mosaic virus, maizechlorotic dwarf virus, cucumber mosaic virus, tobacco mosaic virus,alfalfa mosaic virus, potato virus X, potato virus Y, potato leaf rollvirus and tomato golden mosaic virus.

Military and Bioterrorism Applications:

In yet other embodiments, the engineered microbe-targeting molecules andproduct comprising thereof can be used to detect or combat bioterroragents (e.g., B. Anthracis, and smallpox).

In accordance with some embodiments described herein, an engineeredmicrobe-binding molecule or microbe-binding substrate can be modified tobind to any of the microbes, e.g., the ones described herein, includingthe associated microbial matter (e.g., but not limited to, fragments ofcell wall, microbial nucleic acid and endotoxin).

Embodiments of the various aspects described herein can be illustratedby the following numbered paragraphs.

-   1. An engineered microbe-targeting molecule comprising:    -   a. at least one microbe surface-binding domain;    -   b. a substrate-binding domain adapted for orienting the microbe        surface-binding domain away from a substrate; and    -   c. at least one linker between the microbe surface-binding        domain and the substrate-binding domain.-   2. The engineered molecule of paragraph 1, wherein the    microbe-surface binding domain comprises a carbohydrate recognition    domain (CRD) or a fragment thereof.-   3. The engineered molecule of paragraph 1 or 2, wherein the CRD or a    fragment thereof further comprises at least a portion of a    carbohydrate-binding protein.-   4. The engineered molecule of paragraph 3, wherein the portion of    the carbohydrate-binding protein excludes at least one of complement    and coagulation activation region.-   5. The engineered molecule of any of paragraphs 2-4, wherein the CRD    or the carbohydrate-binding protein is derived from a lectin, a    ficolin, or a fragment thereof.-   6. The engineered molecule of paragraph 5 wherein the lectin is    C-type lectin, or a fragment thereof.-   7. The engineered molecule of paragraph 6, wherein the C-type lectin    is collectin, or a fragment thereof.-   8. The engineered molecule of paragraph 7, wherein the collectin is    mannose-binding lectin (MBL) or a fragment thereof.-   9. The engineered molecule of any of paragraphs 2-8, wherein the CRD    is of SEQ ID NO. 4 or a fragment thereof.-   10. The engineered molecule of any of paragraphs 2-9, wherein the    CRD or a fragment thereof further comprises a neck region of the    carbohydrate-binding protein or a fragment thereof.-   11. The engineered molecule of any of paragraphs 1-10, wherein the    substrate-binding domain comprises at least one amine.-   12. The engineered molecule of any of paragraphs 1-11, wherein the    substrate-binding domain comprises at least one oligopeptide    comprising an amino acid sequence of AKT.-   13. The engineered molecule of any of paragraphs 1-12, wherein the    linker is adapted to provide flexibility and orientation of the    carbohydrate recognition domain to bind to the microbe surface.-   14. The engineered molecule of any of paragraphs 1-13, wherein the    linker is adapted to facilitate expression and purification.-   15. The engineered molecule of any of paragraphs 1-14, wherein the    linker comprises a portion of a Fc region of an immunoglobulin.-   16. The engineered molecule of paragraph 15, wherein the    immunoglobulin is selected from the group consisting of IgA, IgD,    IgE, IgG, and IgM.-   17. The engineered molecule of paragraph 15 or 16, wherein the    immunoglobulin is IgG1.-   18. The engineered molecule of any of paragraphs 15-17, wherein the    portion of the Fc region comprises at least one region selected from    the group consisting of a hinge region, a CH2 region, a CH3 region,    and any combinations thereof.-   19. The engineered molecule of any of paragraphs 15-18, wherein the    portion of the Fc region comprises at least one hinge region, at    least one CH2 region and at least one CH3 region.-   20. The engineered molecule of any of paragraphs 15-19, wherein the    portion of the Fc region comprises at least one mutation.-   21. The engineered molecule of paragraph 20, wherein the at least    one mutation is selected to increase half-life of the engineered    molecule.-   22. The engineered molecule of any of paragraphs 20-21, wherein the    mutation is selected to modulate antibody-dependent cell-mediated    cytotoxicity.-   23. The engineered molecule of any of paragraphs 20-22, wherein the    mutation is selected to modulate complement-dependent cytotoxicity.-   24. The engineered molecule of any of paragraphs 20-23, wherein the    mutation occurs at amino acid residue 82 of SEQ ID NO. 9 from    asparagine to aspartic acid.-   25. The engineered molecule of any of paragraphs 15-24, wherein    N-terminus of the Fc region is adapted for linking to the    substrate-binding domain.-   26. The engineered molecule of any of paragraphs 1-25, wherein the    linker is part of the carbohydrate-binding protein, the neck region,    the Fc region, or any combinations thereof.-   27. The engineered molecule of any of paragraphs 1-26, wherein the    engineered molecule is a dimer.-   28. The engineered molecule of paragraph 27, wherein the dimer is    formed by dimerizing the Fc region of two engineered molecules.-   29. The engineered molecule of any of paragraphs 1-28, further    comprising a detectable label.-   30. The engineered molecule of paragraph 29, wherein the detectable    label is selected from the group consisting of biotin, a fluorescent    dye or particle, a luminescent or bioluminescent marker, a    radiolabel, an enzyme, a microbial enzyme substrate, a quantum dot,    an imaging agent, and any combinations thereof.-   31. The engineered molecule of paragraph 30, wherein the enzyme    causes a color change in the presence of an enzyme substrate.-   32. The engineered molecule of paragraph 31, wherein the enzyme is a    horseradish peroxidase or alkaline phosphatase.-   33. An engineered mannose-binding lectin molecule comprising:    -   a. at least one carbohydrate recognition domain (CRD) or a        fragment thereof;    -   b. a substrate-binding domain adapted for orienting the CRD away        from a substrate; and    -   c. at least one linker between the CRD and the substrate-binding        domain.-   34. The engineered lectin of paragraph 33, wherein the CRD is of SEQ    ID NO. 4.-   35. The engineered lectin of paragraph 33 or 34, wherein the CRD or    a fragment thereof further comprises at least a portion of    mannose-binding lectin (MBL).-   36. The engineered lectin of any of paragraphs 33-35, wherein the    portion of the MBL excludes at least one of complement and    coagulation activation region.-   37. The engineered lectin of any of paragraphs 33-36, wherein the    CRD further comprises a neck region of the MBL.-   38. The engineered lectin of any of paragraphs 33-37, wherein the    substrate-binding domain comprises at least one amine.-   39. The engineered lectin of any of paragraphs 33-38, wherein the    substrate-binding domain comprises at least one oligopeptide    comprising an amino acid sequence of AKT.-   40. The engineered lectin of any of paragraphs 33-39, wherein the    linker is adapted to provide flexibility and orientation of the    carbohydrate recognition domain to bind to the microbe surface.-   41. The engineered lectin of any of paragraphs 33-40, wherein the    linker is adapted to facilitate expression and purification.-   42. The engineered lectin of any of paragraphs 33-41, wherein the    linker comprises a portion of a Fc region of an immunoglobulin.-   43. The engineered lectin of paragraph 42, wherein the    immunoglobulin is selected from the group consisting of IgA, IgD,    IgE, IgG, and IgM.-   44. The engineered lectin of paragraph 42 or 43, wherein the    immunoglobulin is IgG1.-   45. The engineered lectin of any of paragraphs 42-44, wherein the    portion of the Fc region comprises at least one region selected from    the group consisting of a hinge region, a CH2 region, a CH3 region,    and any combinations thereof.-   46. The engineered lectin of any of paragraphs 42-45, wherein the    portion of the Fc region comprises at least one hinge region, at    least one CH2 region and at least one CH3 region.-   47. The engineered lectin of any of paragraphs 42-46, wherein the    portion of the Fc region comprises at least one mutation.-   48. The engineered lectin of paragraph 47, wherein the mutation is    selected to increase half-life of the engineered molecule.-   49. The engineered lectin of paragraph 48, wherein the mutation    occurs at an amino acid residue 232 of SEQ ID NO. 9 from lysine to    alanine.-   50. The engineered lectin of any of paragraphs 47-49, wherein the    mutation is selected to modulate antibody-dependent cell-mediated    cytotoxicity.-   51. The engineered lectin of any of paragraphs 47-50, wherein the    mutation is selected to modulate complement-dependent cytotoxicity.-   52. The engineered lectin of any of paragraphs 47-51, wherein the    mutation occurs at amino acid site 82 of SEQ ID NO. 9 from    asparagine to aspartic acid.-   53. The engineered lectin of any of paragraphs 47-52, wherein    N-terminus of the Fc region is adapted for linking to the    substrate-binding domain.-   54. The engineered lectin of any of paragraphs 47-53, wherein the    linker is part of the mannose-binding lectin, the neck region, the    Fc region, or any combinations thereof.-   55. The engineered lectin of any of paragraphs 33-54, wherein the    engineered molecule is a dimer.-   56. The engineered lectin of paragraph 55, wherein the dimer is    formed by dimerizing the Fc region of two engineered lectin    molecules.-   57. The engineered lectin of any of paragraphs 33-56, further    comprising a detectable label.-   58. The engineered lectin of paragraph 57, wherein the detectable    label or imaging agent is selected from the group consisting of    biotin, a fluorescent dye or particle, a luminescent or    bioluminescent marker, a radiolabel, an enzyme, a microbial enzyme    substrate, a quantum dot, an imaging agent, and any combinations    thereof.-   59. The engineered lectin of paragraph 58, wherein the enzyme causes    a color change in the presence of an enzyme substrate.-   60. The engineered lectin of paragraph 59, wherein the enzyme is a    horseradish peroxidase or alkaline phosphatase.-   61. An engineered microbe-targeting molecule comprising:    -   a. at least one microbe surface-binding domain; and    -   b. at least a portion of a Fc region of an immunoglobulin.-   62. The engineered molecule of paragraph 61, wherein the portion of    the Fc region is linked to N-terminal of the microbe surface-binding    domain.-   63. The engineered molecule of paragraph 61 or 62, wherein the    microbe surface-binding domain comprises a carbohydrate recognition    domain (CRD) or a fragment thereof.-   64. The engineered molecule of paragraph 63, wherein the CRD or a    fragment thereof further comprises at least a portion of a    carbohydrate-binding protein.-   65. The engineered molecule of paragraph 64, wherein the portion of    the carbohydrate-binding protein excludes at least one of complement    and coagulation activation region.-   66. The engineered molecule of any of paragraphs 63-65, wherein the    CRD or the carbohydrate-binding protein is derived from a lectin, a    ficolin, or a fragment thereof.-   67. The engineered molecule of paragraph 66, wherein the lectin is    C-type lectin, or a fragment thereof.-   68. The engineered molecule of paragraph 67, wherein the C-type    lectin is collectin, or a fragment thereof.-   69. The engineered molecule of paragraph 68, wherein the collectin    is mannose-binding lectin (MBL) or a fragment thereof.-   70. The engineered molecule of any of paragraphs 63-69, wherein the    CRD is of SEQ ID NO. 4 or a fragment thereof.-   71. The engineered molecule of any of paragraphs 63-70, wherein the    CRD or a fragment thereof further comprises a neck region of a    carbohydrate-binding protein.-   72. The engineered molecule of any of paragraphs 61-71, wherein said    at least a portion of the Fc region of the immunoglobulin further    comprises a substrate-binding domain.-   73. The engineered molecule of paragraph 72, wherein the    substrate-binding domain comprises at least one amine.-   74. The engineered molecule of any of paragraphs 61-73, wherein the    substrate-binding domain comprises at least one oligopeptide    comprising an amino acid sequence of AKT.-   75. The engineered molecule of any of paragraphs 61-74, wherein the    immunoglobulin is selected from the group consisting of IgA, IgD,    IgE, IgG, and IgM.-   76. The engineered molecule of any of paragraphs 61-75, wherein the    immunoglobulin is IgG1.-   77. The engineered molecule of any of paragraphs 61-76, wherein the    portion of the Fc region comprises at least one region selected from    the group consisting of a hinge region, a CH2 region, a CH3 region,    and any combinations thereof.-   78. The engineered molecule of any of paragraphs 61-77, wherein the    portion of the Fc region comprises at least one hinge region, at    least one CH2 region and at least one CH3 region.-   79. The engineered molecule of any of paragraphs 61-78, wherein the    portion of the Fc region comprises at least one mutation.-   80. The engineered molecule of paragraph 79, wherein the at least    one mutation is selected to increase half-life of the engineered    microbe-binding molecule.-   81. The engineered molecule of any of paragraphs 61-80, wherein the    mutation is selected to modulate antibody-dependent cell-mediated    cytotoxicity.-   82. The engineered molecule of any of paragraphs 61-81, wherein the    mutation is selected to modulate complement-dependent cytotoxicity.-   83. The engineered molecule of any of paragraphs 61-82, wherein the    mutation occurs at amino acid residue 82 of SEQ ID NO. 9 from    asparagine to aspartic acid.-   84. The engineered molecule of any of paragraphs 61-83, wherein the    engineered molecule is a dimer.-   85. The engineered molecule of paragraph 84, wherein the dimer is    formed by dimerizing the Fc region of two engineered molecules.-   86. The engineered molecule of any of paragraphs 61-85, further    comprising a detectable label.-   87. The engineered molecule of paragraph 86, wherein the detectable    label is selected from the group consisting of biotin, a fluorescent    dye or particle, a luminescent or bioluminescent marker, a    radiolabel, an enzyme, a microbial enzyme substrate, a quantum dot,    an imaging agent, and any combinations thereof.-   88. The engineered molecule of paragraph 87, wherein the enzyme    causes a color change in the presence of an enzyme substrate.-   89. The engineered molecule of paragraph 88, wherein the enzyme is a    horseradish peroxidase or alkaline phosphatase.-   90. A microbe-targeting substrate or a product comprising a    substrate, and at least one engineered microbe-targeting molecule of    any of paragraphs 1-32 and 61-89 or at least one engineered    mannose-binding lectin molecule of any of paragraphs 33-60, wherein    the substrate comprises on its surface said at least one engineered    microbe-targeting molecule or at least one engineered    mannose-binding lectin molecule.-   91. The microbe-targeting substrate or the product of paragraph 90,    wherein the substrate-binding domain of the engineered    microbe-targeting molecule or mannose-binding lectin molecule is    adapted for binding to the substrate.-   92. The microbe-targeting substrate or the product of paragraph 90    or 91, wherein the substrate is selected from the group consisting    of a nucleic acid scaffold, a protein scaffold, a lipid scaffold, a    dendrimer, microparticle or a microbead, a nanotube, a microtiter    plate, a medical apparatus or implant, a microchip, a filtration    device, a membrane, a diagnostic strip, a dipstick, an    extracorporeal device, a spiral mixer, and a hollow-fiber reactor.-   93. The microbe-targeting substrate or the product of any of    paragraphs 90-92, wherein the substrate is a microparticle.-   94. The microbe-targeting substrate or the product of paragraph 93,    wherein the microparticle is a magnetic microparticle.-   95. The microbe-targeting substrate or the product of paragraph 93,    wherein the microparticle is a fluorescent microparticle or a    quantum dot.-   96. The microbe-targeting substrate or the product of paragraph 93,    wherein the microparticle is a drug delivery vehicle.-   97. The microbe-targeting substrate or the product of any of    paragraphs 90-96, wherein the substrate is a dipstick.-   98. The microbe-targeting substrate or the product of any of    paragraphs 90-96, wherein the substrate is a membrane.-   99. The microbe-targeting substrate or the product of paragraph 97    or 98, wherein the dipstick or the membrane comprises on its surface    at least an area adapted for use as a reference area.-   100. The microbe-targeting substrate or the product of any of    paragraphs 90-99, wherein the substrate is a living cell, or a    biological tissue or organ.-   101. The microbe-targeting substrate or the product of any of    paragraphs 90-100, wherein the substrate is functionalized.-   102. The microbe-targeting substrate or the product of any of    paragraphs 90-101, wherein the substrate is treated to become less    adhesive to a biological molecule.-   103. The microbe-targeting substrate or the product of paragraph    102, wherein the biological molecule is selected from the group    consisting of blood cells and components, proteins, nucleic acids,    peptides, small molecules, therapeutic agents, cells or fragments    thereof, and any combinations thereof.-   104. A pharmaceutical composition comprising at least one engineered    microbe-targeting molecule of any of paragraphs 1-32 and 61-89 or at    least one engineered mannose-binding lectin molecule of any of    paragraphs 33-60 or at least one microbe-targeting substrate of any    of paragraphs 90-103, and a pharmaceutically acceptable carrier.-   105. A kit comprising:    -   a. one or more containers containing a population of engineered        microbe-targeting molecules of any of paragraphs 1-32 and 61-89        or a population of engineered mannose-binding lectin molecules        of any of paragraphs 33-60; and    -   b. at least one reagent.-   106. The kit of paragraph 105, further comprising one or more    substrates to which the engineered microbe-targeting molecules or    engineered mannose-binding lectin molecules are conjugated.-   107. The kit of paragraph 105 or 106, wherein the substrates are    selected from the group consisting of a nucleic acid scaffold, a    protein scaffold, a lipid scaffold, a dendrimer, microparticle or a    microbead, a nanotube, a microtiter plate, a medical apparatus or    implant, a microchip, a filtration device, a membrane, a diagnostic    strip, a dipstick, an extracorporeal device, a spiral mixer, and a    hollow-fiber reactor.-   108. The kit of any of paragraphs 105-107, wherein the substrates    include a population of the microbeads.-   109. The kit of paragraph 108, wherein the microbeads are magnetic    microbeads.-   110. A kit comprising:    -   a. one or more microbe-targeting substrates of any of paragraphs        90-104; and    -   b. at least one reagent.-   111. The kit of any of paragraphs 105-108, wherein the one or more    microbe-targeting substrates include dipsticks.-   112. The kit of any of paragraphs 105-109, wherein the one or more    microbe-targeting substrates include a population of    microbe-targeting microbeads.-   113. The kit of paragraph 108, 109 or 112, wherein the population of    microbes or microbe-targeting microbeads is provided in one or more    separate containers.-   114. The kit of any of paragraphs 108, 109, and 112-113, wherein the    population of the microbeads or microbe-targeting microbeads    comprises at least one distinct subset, the distinct subset    comprising microbeads or microbe-targeting microbeads having a    dimension different from the rest of the population.-   115. The kit of any of paragraphs 110-114, wherein the    microbe-targeting microbeads each further comprises a detection    label.-   116. The kit of any of paragraphs 105-115, further comprising one or    more containers each containing a population of detectable labels,    wherein each of the detectable label is conjugated to a molecule.-   117. The kit of paragraph 116, wherein at least one of the    containers contains a distinct population of detectable labels.-   118. The kit of any of paragraphs 116-117, wherein the molecule is    an engineered microbe-targeting molecule of any of paragraphs 1-32    and 61-89 or an engineered mannose-binding lectin molecules of any    of paragraphs 33-60.-   119. The kit of paragraph 118, wherein the molecule comprises at    least a carbohydrate recognition domain (CRD) or a fragment thereof.-   120. The kit of paragraph 119, wherein at least one population of    the molecule comprises SEQ ID NO. 4 or a fragment thereof.-   121. The kit of any of paragraphs 116-120, wherein the molecule    further comprises a Fc region of an immunoglobulin.-   122. The kit of any of paragraphs 116-120, wherein the molecule    includes an antibody specific to the microbe.-   123. The kit of any of paragraphs 116-122, wherein the detectable    label comprises an enzyme that produces a color change in the    presence of an enzyme substrate.-   124. The kit of paragraph 123, wherein the enzyme is a horseradish    peroxidase, an alkaline phosphatase, or any combinations thereof.-   125. The kit of any of paragraphs 105-124, further comprising one or    more containers containing an enzyme substrate that changes color in    the presence of the enzyme.-   126. The kit of any of paragraphs 116-125, wherein the detectable    label comprises a fluorescent molecule.-   127. The kit of any of paragraphs 105-126, wherein the at least one    reagent is a wash buffer, a dilution buffer, a stop buffer, a    buffered solution containing a chelating agent, a coupling agent    used for conjugation of the engineered molecule to the substrate, or    any combinations thereof.-   128. The kit of any of paragraphs 105-127, further comprising at    least one microtiter plate.-   129. The kit of any of paragraphs 108-128, wherein the population of    microbeads or microbe-targeting microbeads is lyophilized.-   130. The kit of any of paragraphs 105-129, further comprising at    least one blood collection container.-   131. The kit of paragraph 130, wherein the population of the    microbe-targeting microbeads is pre-loaded into said at least one    blood collection container.-   132. The kit of paragraph 130 or 131, wherein the blood collection    container further comprises an anti-coagulant agent.-   133. The kit of any of paragraphs 112-132, wherein the engineered    microbe-targeting microbeads are microbe-targeting magnetic    microbeads.-   134. The kit of paragraph 133, further comprising a magnet adapted    for collecting the microbe-targeting magnetic microbeads in the    blood collection container.-   135. The kit of any of paragraphs 105-134, further comprising a    reference for comparison with a readout determined from a test    sample.-   136. The kit of any of paragraphs 110-135, wherein one or more    microbe-targeting substrates are individually packaged.-   137. A method of detaching a microbe and/or microbial matter from a    microbe-targeting molecule, the method comprising incubating the    substrate with buffer having an acidic pH.-   138. The method of paragraph 137, wherein the buffer has a pH about    6.5 or lower.-   139. The method of paragraph 137 or 138, wherein the buffer    comprises 0.2M glycine and has a pH of about 2.8.-   140. A method of detaching a microbe and/or microbial matter from a    microbe-targeting molecule, the method comprising incubating the    substrate with a buffer comprising an ion which forms a salt with    Ca2+ ion and wherein the said salt is insoluble in the buffer.-   141. The method of paragraph 140, wherein said ion is selected from    the group consisting of phosphate, oxalate, carbonate, sulfate,    fluoride, gluconic acid, oxido-trioxo-manganese, stearic acid, and    any combinations thereof.-   142. The method of paragraph 140 or 141, wherein said ion is present    at a concentration of about 0.05M to about 5M.-   143. The method of any of paragraphs 140-142, wherein the buffer    comprises about 0.1M sodium phosphate and has pH of about 6.8.-   144. The method of any of paragraphs 140-143, wherein the    interaction between the microbe and the microbe-targeting molecule    is mediated by a Ca2+ ion.-   145. The method of any of paragraphs 140-144, wherein the aqueous    solution further comprises a chelating agent.-   146. The method of paragraph 145, wherein the chelating agent is    selected from the group consisting of    1,2-bis(2-Aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid;    ethylenediaminetetraacetic acid (EDTA); ethylene    glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA);    ethylene glycol-bis(β-aminoethyl ether)-N,N,N′, N′-tetraacetic acid,    1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA),    nitrile-2,2′,2″-triacetic acid (NTA), and any combinations thereof.-   147. The method of any of paragraphs 137-146, wherein the substrate    is microparticle.-   148. The method of any of paragraphs 137-147, wherein the substrate    is a magnetic microparticle.-   149. The method of any of paragraphs 137-148, wherein the    microbe-targeting molecule is an engineered microbe-targeting    molecule of any of paragraphs 1-32 or 61-89, or an engineered    mannose-binding ligand of any of paragraphs 33-60.-   150. The method of any of paragraphs 137-149, further comprising    heating or cooling the buffer during said contacting.-   151. The method of any of paragraphs 137-150, further comprising    shaking the substrate in the buffer.-   152. The method of any of paragraphs 137-151, wherein said    incubation is for at least 5 minutes.-   153. The method of any of paragraphs 137-152, further comprising    washing the substrate with after detachment of the microbe.-   154. The method of any of paragraphs 137-153, wherein the    microbe-targeting molecule binds to a substrate.-   155. A composition for treating and/or preventing a microbial    infection or a microbial contamination comprising at least one    engineered microbe-targeting molecule of any of paragraphs 1-32 or    61-89 or at least one engineered mannose-binding lectin molecule of    any of paragraphs 33-60 or at least one microbe-targeting substrate    of any of paragraphs 90-103.-   156. The composition of paragraph 155, wherein the composition is    formulated for treating and/or preventing a microbial infection or a    microbial contamination present in an environment surface.-   157. The composition of paragraph 156, wherein the environmental    surface includes a medical device, an implantable device, a surface    in a hospital or clinic (e.g., an operating room or an    intensive-care unit), a machine or working surface for manufacturing    or processing food or pharmaceutical products, a cell culture, a    water treatment plant, a water reservoir or a botanical plant.-   158. The composition of any of paragraphs 155-157, wherein the    composition is formulated for treating and/or preventing a microbial    infection in a body fluid of a subject.-   159. The composition of any of paragraphs of 155-158, wherein the    composition is formulated for treating and/or preventing a microbial    infection in a tissue of a subject.-   160. The composition of paragraph 158 or 159, wherein the subject is    a mammalian subject.-   161. The composition of any of paragraphs 155-160, wherein said at    least one engineered microbe-targeting molecule is present in an    amount effective to reduce the growth and/or spread of the microbe.-   162. The composition of any of paragraphs 155-161, further    comprising at least one of an antimicrobial agent and a drug    delivery vehicle.-   163. The composition of paragraph 162, wherein at least one of the    engineered microbe-targeting molecule and the antimicrobial agent is    coated on a surface of the drug delivery vehicle.-   164. The composition of paragraph 162 or 163, wherein the drug    delivery vehicle is selected from the group consisting of a peptide    particle, a polymeric particle, a dendrimer, a vesicle, a liposome,    a hydrogel, a nucleic acid scaffold, an aptamer, and any    combinations thereof,-   165. The composition of any of paragraphs 162-164, wherein the    antimicrobial agent is fused with said at least one engineered    microbe-targeting molecule.-   166. The composition of any of paragraphs 162-165, wherein the    antimicrobial agent is selected from the group consisting of silver    nanoparticle, an antimicrobial metalloendopeptidase, an    antimicrobial peptide, an antibiotic, and any combinations thereof.-   167. The composition of any of paragraphs 155-166, wherein a microbe    causing the microbial infection or microbial contamination is a    protein A-expressing microbe, a protein G-expressing microbe or any    combinations thereof.-   168. The composition of paragraph 167, wherein the protein    A-expressing microbe includes Staphylococcus or the protein    G-expressing microbe includes Streptococcus.-   169. The composition of paragraph 167 or 168, wherein the protein    A-expressing microbe includes Staphylococcus aureus.-   170. The composition of any of paragraphs 167-169, wherein the    microbe is resistant to at least one antimicrobial agent.-   171. The composition of paragraph 170, wherein the antimicrobial    agent is an antibiotic.-   172. The composition of paragraph 171, wherein the antibiotic is    selected from the group consisting of aminoglycosides, ansamycins,    carbacephem, carbapenems, cephalosporins, glycopeptides,    lincosamides, lipopeptide, macrolides, monobactams, nitrofurans,    penicillins, polypeptides, quinolones, sulfonamides, tetracyclines,    methicillin, vancomycin, and any combinations thereof.-   173. The composition of any of paragraphs 167-172, wherein the    protein A-expressing microbe includes methicillin-resistant    Staphylococcus aureus.-   174. The composition of any of paragraphs 167-173, wherein the    protein A-expressing microbe includes vancomycin-resistant    Staphylococcus aureus.-   175. The composition of any of paragraphs 155-174, wherein the    composition is adapted for use as a wound dressing.-   176. The composition of any of paragraphs 155-175, wherein the    immunoglobulin is a human immunoglobulin.-   177. A method for removing a microbe and/or microbial matter from a    target area comprising contacting the target area with a composition    of any of paragraphs 155-176.-   178. A method for treating and/or preventing a microbial infection    or microbial contamination in a target area comprising contacting    the target area with a first composition of any of paragraphs    155-176.-   179. The method of paragraph 177 or 178, wherein the target area    includes an environmental surface.-   180. The method of paragraph 179, wherein the environmental surface    includes a medical device, an implantable device, a surface in a    hospital or clinic (e.g., an operating room or an intensive-care    unit), a machine or working surface for manufacturing or processing    food or pharmaceutical products, a cell culture, a water treatment    plant, a water reservoir or a botanical plant.-   181. The method of paragraph 177 or 178, wherein the target area is    present in a body fluid of a subject.-   182. The method of paragraph 177 or 178, wherein the target area is    present in a tissue of a subject.-   183. The method of paragraph 182, further comprising replacing the    first composition in contact with the tissue with a second    composition of any of paragraphs 157-178 after a period of time.-   184. The method of paragraph 182 or 183, further comprising    administering an additional treatment to the tissue.-   185. The method of paragraph 184, wherein the additional treatment    includes a negative-pressure treatment, a vacuum-assisted    debridement, administration of an antimicrobial agent, or any    combinations thereof.-   186. An assay for determining the presence or absence of a microbe    and/or microbial matter in a test sample, the assay comprising:    contacting a test sample with a microbe-targeting substrate of any    of paragraphs 90-103.-   187. An assay of determining the presence or absence of a microbe    and/or microbial matter in a test sample, the method comprising:    contacting a test sample with a plurality of microbe-targeting    substrates of any of paragraphs 90-103, wherein the plurality of    microbe-targeting substrates comprises a first subset of    microbe-targeting substrates and a second subset of    microbe-targeting substrates; and    -   wherein the first subset of microbe-targeting substrates each        has a first pre-determined dimension; and    -   wherein the second subset of microbe-targeting substrates each        has a second pre-determined dimension.-   188. The assay of paragraph 187, wherein the first subset and the    second subset are added to the test sample to form a single mixture.-   189. The assay of paragraph 187, wherein the second subset is added    to the test sample after isolation of the first subset previously    added to the test sample.-   190. The assay of any of paragraph 186-189, wherein the    microbe-targeting substrate is in a form of a microbead.-   191. The assay of paragraph 190, wherein the first pre-determined    dimension and the second pre-determined dimension of the microbead    range from about 10 nm to about 10 μm.-   192. The assay of paragraph 190, wherein the first pre-determined    dimension and the second pre-determined dimension of the microbead    range from about 50 nm to about 200 nm.-   193. The assay of any of paragraphs 190-192, wherein the microbead    is a magnetic microbead.-   194. The assay of any of paragraphs 186-193, further comprising    analyzing the microbe-targeting substrate for the presence or    absence of a bound microbe and/or microbial matter, wherein the    presence of a microbe-targeting substrate-bound microbe and/or    microbial matter indicates that the test sample is infected with a    microbe; and the absence of a microbe-targeting substrate-bound    microbe and/or microbial matter indicates the test sample contains    no detectable microbes or microbial matter.-   195. The assay of any of paragraphs 186-194, wherein the microbial    matter includes endotoxin.-   196. An assay for determining the presence or absence of a protein-A    expressing microbe, a protein-G expressing microbe, or microbial    matter thereof, in a test sample, the assay comprising:    -   contacting a test sample with a microbe-targeting substrate of        any of paragraphs 90 to 103 in the presence of a chelating        agent.-   197. The assay of paragraph 196, further comprising analyzing the    microbe-targeting substrate for the presence or absence of a bound    microbe, wherein the presence of a microbe-targeting substrate-bound    microbe indicates the presence of a protein-A expressing microbe or    a protein G-expressing microbe in the test sample; and the absence    of a microbe-targeting substrate-bound microbe indicates the absence    of a protein-A expressing or a protein G-expressing microbe in the    test sample.-   198. The assay of paragraph 197, wherein in the absence of a    microbe-targeting substrate-bound microbe, the test sample is    further contacted with the microbe-targeting substrate in the    presence of free calcium ions.-   199. An assay for detecting a protein-A expressing microbe, a    protein-G expressing microbe, or microbial matter thereof, in a test    sample, the assay comprising:    -   i. contacting a test sample with a microbe-targeting substrate        of any of paragraphs 90 to 103;    -   ii. contacting the microbe-binding molecule with a solution        comprising a chelating agent; and    -   iii. analyzing the microbe-targeting substrate for the presence        or absence of a bound microbe, wherein the presence of a        microbe-targeting substrate-bound microbe indicates the presence        of a protein A-expressing microbe or a protein G-expressing        microbe in the test sample; and the absence of a        microbe-targeting substrate-bound microbe indicates the absence        of a protein A-expressing microbe or a protein G-expressing        microbe in the test sample.-   200. The assay of paragraph 199, further comprising isolating the    microbe-targeting substrate from the test sample before contacting    with the solution comprising the chelating agent.-   201. The assay of any of paragraphs 186-200, further comprising    isolating the microbe-targeting substrate from the test sample or    the solution comprising the chelating agent before the analyzing    step.-   202. The assay of paragraph 201, wherein the analyzing comprises an    immunoassay, ELISA, Gram staining, immunostaining, microscopy,    spectroscopy, immunofluorescence, western blot, PCR, RT-PCR,    fluorescence in situ hybridization, sequencing, mass spectroscopy,    and any combinations thereof.-   203. The assay of any of paragraphs 186-202, further comprising    culturing the microbe bound on the microbe-targeting substrate.-   204. The assay of any of paragraphs 186-203, further comprising    subjecting the microbe bound on the microbe-targeting substrate to    an antibiotic.-   205. The assay of any of paragraphs 186-204, wherein the    microbe-targeting substrate is preformed from at least a substrate    and said at least one engineered microbe-binding molecule before the    contacting.-   206. The assay of any of paragraphs 186-204, wherein the    microbe-targeting substrate is formed from at least said substrate    and said at least one engineered microbe-binding molecule during the    contacting.-   207. The assay of any of paragraphs 196-206, wherein the presence of    the chelating agent reduces the likelihood of a protein A- and    protein G-negative microbe, if present, in the test sample, to bind    with said at least one engineered microbe-binding molecule.-   208. The assay of any of paragraphs 186-207, further comprising    detaching the bound microbe from the microbe-targeting substrate.-   209. The assay of paragraph 208, further comprising contacting the    isolated microbe-targeting substrate with a low pH buffer.-   210. The assay of any of paragraphs 196-209, wherein the chelating    agent is a metal-ion chelating agent.-   211. The assay of any of paragraphs 196-210, wherein the chelating    agent chelates a calcium ion.-   212. The assay of paragraph 211, wherein the calcium-chelating agent    is selected from the group consisting of    1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid,    ethylenediaminetetraacetic acid (EDTA); ethylene    glycol-bis(2-aminoethylether)-N,N,N′, N′-tetraacetic acid; ethylene    glycol-bis(□-aminoethyl ether)-N,N,N?,N?-tetraacetic acid (EGTA),    1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), a    buffer containing citrate,    N,N-Bis(2-(bis-(carboxymethyl)amino)ethyl)-glycine (DTPA),    nitrilo-2,2′,2″-triacetic acid (NTA), a buffer that precipitates a    calcium ion from the test sample, a low pH buffer, any derivatives    thereof, and any combinations thereof.-   213. The assay of any of paragraphs 209-212, wherein the low pH    buffer has a pH less than 7.-   214. The assay of any of paragraphs 209-213, wherein the low pH    buffer is selected from the group consisting of arginine and    pyrophosphate.-   215. The assay of any of paragraphs 196-214, wherein the protein    A-expressing microbe includes Staphylococcus, or the protein    G-expressing microbe includes Streptococcus.-   216. The assay of paragraph 215, wherein the protein A-expressing    microbe includes Staphylococcus aureus.-   217. The assay of paragraph 215, wherein the Staphylococcus species    excludes Staphylococcus epidermidis.-   218. The assay of any of paragraphs 186-217, further comprising    analyzing at least one microbe-targeting substrate upon contact with    the test sample before contacting the microbe-binding molecule with    the solution comprising the chelating agent.-   219. The assay of paragraph 186-218, wherein the microbe-targeting    substrate is in a form of a microbead.-   220. The assay of paragraph 219, wherein the microbead is a magnetic    microbead.-   221. A method of determining the presence or absence of    Staphylococcus aureus infection in a subject, comprising performing    the assay of any of paragraphs 190-214, wherein the binding of a    microbe to said at least one engineered microbe-targeting substrate    in the presence of a chelating agent is indicative of Staphylococcus    aureus infection in the subject.-   222. The method of paragraph 221, further comprising administering    or prescribing to the subject a first antimicrobial agent when the    subject is detected with Staphylococcus aureus.-   223. The method of paragraph 221 or 222, further comprising    analyzing the test sample or the solution comprising the chelating    agent after isolating the engineered microbe-targeting substrate    therefrom to determine the presence or absence of a protein    A-negative or a protein G-negative microbe.-   224. The method of paragraph 223, further comprising administering    or prescribing to the subject a second antimicrobial agent when the    subject is detected with a protein A-negative or a protein    G-negative microbe.-   225. The method of paragraph 224, wherein the protein A-negative or    the protein G-negative microbe include E. coli.-   226. The method of any of paragraphs 221-225, further comprising    administering or prescribing to the subject a composition comprising    at least one engineered microbe-targeting molecule of any of    paragraphs 1-32 or 61-89, or at least one engineered mannose-binding    lectin molecule of any of paragraphs 33-60.

Some Selected Definitions

Unless stated otherwise, or implicit from context, the following termsand phrases include the meanings provided below. Unless explicitlystated otherwise, or apparent from context, the terms and phrases belowdo not exclude the meaning that the term or phrase has acquired in theart to which it pertains. The definitions are provided to aid indescribing particular embodiments of the aspects described herein, andare not intended to limit the claimed invention, because the scope ofthe invention is limited only by the claims. Further, unless otherwiserequired by context, singular terms shall include pluralities and pluralterms shall include the singular.

As used herein the term “comprising” or “comprises” is used in referenceto compositions, methods, and respective component(s) thereof, that areessential to the invention, yet open to the inclusion of unspecifiedelements, whether essential or not.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof additional elements that do not materially affect the basic and novelor functional characteristic(s) of that embodiment of the invention.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages maymean±1%.

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. Thus for example, references to “the method” includes one ormore methods, and/or steps of the type described herein and/or whichwill become apparent to those persons skilled in the art upon readingthis disclosure and so forth.

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of this disclosure,suitable methods and materials are described below. The term “comprises”means “includes.” The abbreviation, “e.g.” is derived from the Latinexempli gratia, and is used herein to indicate a non-limiting example.Thus, the abbreviation “e.g.” is synonymous with the term “for example.”

The terms “microbe-binding” and “microbe-targeting” as usedinterchangeably herein refers to an ability of a molecule or compositionto bind and/or capture a microbe and/or microbial matter.

The term “FcMBL microbead” as used herein refers to a microbeadcomprising on its surface at least one FcMBL molecule. In someembodiments, the microbead comprises on its surface a saturating amountof the FcMBL molecules. A microbead can be magnetic or non-magnetic.

The term “FcMBL magnetic microbead” as used herein refers to a magneticmicrobead comprising on its surface at least one FcMBL molecule. In someembodiments, the magnetic microbead comprises on its surface asaturating amount of the FcMBL molecules.

The term “antibody” as used herein refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin molecules(molecules that contain an antigen binding site which specifically bindsan antigen), including monoclonal antibodies (including full lengthmonoclonal antibodies), polyclonal antibodies, multispecific antibodies(for example, bispecific antibodies), chimeric antibodies, humanizedantibodies, human antibodies, and single chain antibodies (scFvs).

The term “peptide” refers to a polymer of amino acids, or amino acidanalogs, regardless of its size or function. In some embodiments, theterm “peptide” refers to small polypeptides, e.g., a polymer of about15-25 amino acids.

The term “oligonucleotide” as used herein refers to a short nucleic acidpolymer, typically with twenty or fewer bases.

As used herein, a “subject” means a human or animal. Usually the animalis a vertebrate such as a primate, rodent, domestic animal or gameanimal. Primates include chimpanzees, cynomologous monkeys, spidermonkeys, and macaques, e.g., Rhesus. Rodents include mice, rats,woodchucks, ferrets, rabbits and hamsters. Domestic and game animalsinclude cows, horses, pigs, deer, bison, buffalo, feline species, e.g.,domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g.,chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon.Patient or subject includes any subset of the foregoing, e.g., all ofthe above, but excluding one or more groups or species such as humans,primates or rodents. In certain embodiments of the aspects describedherein, the subject is a mammal, e.g., a primate, e.g., a human. Theterms, “patient” and “subject” are used interchangeably herein.

In some embodiments, the subject is a mammal. The mammal can be a human,non-human primate, mouse, rat, dog, cat, horse, or cow, but are notlimited to these examples. Mammals other than humans can beadvantageously used as subjects that represent animal models ofdisorders.

A subject can be one who has been previously diagnosed with oridentified as suffering from or having a disease or disorder caused byany microbes or pathogens described herein. By way of example only, asubject can be diagnosed with sepsis, inflammatory diseases, orinfections.

The term “therapeutic agents” is art-recognized and refers to anychemical moiety that is a biologically, physiologically, orpharmacologically active substance that acts locally or systemically ina subject. Examples of therapeutic agents, also referred to as “drugs”,are described in well-known literature references such as the MerckIndex, the Physicians Desk Reference, and The Pharmacological Basis ofTherapeutics, and they include, without limitation, medicaments;vitamins; mineral supplements; substances used for the treatment,prevention, diagnosis, cure or mitigation of a disease or illness;substances which affect the structure or function of the body; orpro-drugs, which become biologically active or more active after theyhave been placed in a physiological environment. Various forms of atherapeutic agent may be used which are capable of being released fromthe subject composition into adjacent tissues or fluids uponadministration to a subject. Examples include steroids and esters ofsteroids (e.g., estrogen, progesterone, testosterone, androsterone,cholesterol, norethindrone, digoxigenin, cholic acid, deoxycholic acid,and chenodeoxycholic acid), boron-containing compounds (e.g.,carborane), chemotherapeutic nucleotides, drugs (e.g., antibiotics,antivirals, antifungals), enediynes (e.g., calicheamicins, esperamicins,dynemicin, neocarzinostatin chromophore, and kedarcidin chromophore),heavy metal complexes (e.g., cisplatin), hormone antagonists (e.g.,tamoxifen), non-specific (non-antibody) proteins (e.g., sugaroligomers), oligonucleotides (e.g., antisense oligonucleotides that bindto a target nucleic acid sequence (e.g., mRNA sequence)), peptides,proteins, antibodies, photodynamic agents (e.g., rhodamine 123),radionuclides (e.g., I-131, Re-186, Re-188, Y-90, Bi-212, At-211, Sr-89,Ho-166, Sm-153, Cu-67 and Cu-64), toxins (e.g., ricin), andtranscription-based pharmaceuticals.

As used here in, the term “peptidomimetic” means a peptide-like moleculethat has the activity of the peptide on which it is structurally based.Such peptidomimetics include chemically modified peptides, peptide-likemolecules containing non-naturally occurring amino acids, and peptoids,and have an activity such as the cardiac specificity of the peptide uponwhich the peptidomimetic is derived (see, for example, Goodman and Ro,Peptidomimetics for Drug Design, in “Burger's Medicinal Chemistry andDrug Discovery”, Vol. 1 (ed. M. E. Wolff; John Wiley & Sons 1995), pages803-861).

A variety of peptidomimetics are known in the art and can be encompassedwithin embodiments described herein including, for example, peptide-likemolecules which contain a constrained amino acid, a non-peptidecomponent that mimics peptide secondary structure, or an amide bondisostere. A peptidomimetic that contains a constrained, non-naturallyoccurring amino acid can include, for example, an α-methylated aminoacid; α,α-dialkylglycine or α-aminocycloalkane carboxylic acid; anNα-Cacyclized amino acid; an Nα-methylated amino acid; αβ- or γ-aminocycloalkane carboxylic acid; an α,β-unsaturated amino acid; aβ,β-dimethyl or β-methyl amino acid; αβ-substituted-2,3-methano aminoacid; an N—Cδ or Cα-Cδ cyclized amino acid; a substituted proline oranother amino acid mimetic. A peptidomimetic which mimics peptidesecondary structure can contain, for example, a nonpeptidic β-turnmimic; γ-turn mimic; mimic of β-sheet structure; or mimic of helicalstructure, each of which is well known in the art. A peptidomimetic alsocan be a peptide-like molecule which contains, for example, an amidebond isostere such as a retro-inverso modification; reduced amide bond;methylenethioether or methylene-sulfoxide bond; methylene ether bond;ethylene bond; thioamide bond; transolefin or fluoroolefin bond;1,5-disubstituted tetrazole ring; ketomethylene or fluoroketomethylenebond or another amide isostere. One skilled in the art understands thatthese and other peptidomimetics are encompassed within the meaning ofthe term “peptidomimetic” as used herein.

Methods for identifying a peptidomimetic are well known in the art andinclude, for example, the screening of databases that contain librariesof potential peptidomimetics. For example, the Cambridge StructuralDatabase contains a collection of greater than 300,000 compounds thathave known crystal structures (Allen et al., Acta Crystallogr. SectionB, 35:2331 (1979)). This structural depository is continually updated asnew crystal structures are determined and can be screened for compoundshaving suitable shapes, for example, the same shape as a peptidedescribed herein, as well as potential geometrical and chemicalcomplementarity to a cognate receptor. Where no crystal structure of apeptide described herein is available, a structure can be generatedusing, for example, the program CONCORD (Rusinko et al., J. Chem. Inf.Comput. Sci. 29:251 (1989)). Another database, the Available ChemicalsDirectory (Molecular Design Limited, Informations Systems; San LeandroCalif.), contains about 100,000 compounds that are commerciallyavailable and also can be searched to identify potential peptidomimeticsof a peptide described herein, for example, having specificity for themicrobes.

The terms “homology” as used herein refers to sequence similaritybetween two peptides or between two nucleic acid molecules. Homology canbe determined by comparing a position in each sequence which may bealigned for purposes of comparison. When an equivalent position in thecompared sequences is occupied by the same base or amino acid, then themolecules are identical at that position; when the equivalent siteoccupied by the same or a similar amino acid residue (e.g., similar insteric and/or electronic nature), then the molecules can be referred toas homologous (similar) at that position. Expression as a percentage ofhomology refers to a function of the number of identical or similaramino acids at positions shared by the compared sequences. A sequencewhich is “unrelated” or “non-homologous” shares less than 40% identity.Determination of homologs of the genes or peptides described herein maybe easily ascertained by the skilled artisan.

The term “conservative substitution,” when describing a polypeptide,refers to a change in the amino acid composition of the polypeptide thatdoes not substantially alter the polypeptide's activity, fore examples,a conservative substitution refers to substituting an amino acid residuefor a different amino acid residue that has similar chemical properties.Conservative amino acid substitutions include replacement of a leucinewith an isoleucine or valine, an aspartate with a glutamate, or athreonine with a serine. “Conservative amino acid substitutions” resultfrom replacing one amino acid with another having similar structuraland/or chemical properties, such as the replacement of a leucine with anisoleucine or valine, an aspartate with a glutamate, or a threonine witha serine. Thus, a “conservative substitution” of a particular amino acidsequence refers to substitution of those amino acids that are notcritical for polypeptide activity or substitution of amino acids withother amino acids having similar properties (e.g., acidic, basic,positively or negatively charged, polar or non-polar, etc.) such thatthe substitution of even critical amino acids does not substantiallyalter activity. Conservative substitution tables providing functionallysimilar amino acids are well known in the art. For example, thefollowing six groups each contain amino acids that are conservativesubstitutions for one another: 1) Alanine (A), Serine (S), Threonine(T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N),Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine(L), Methionine (M), Valine (V); and 6)Phenylalanine (F), Tyrosine (Y),Tryptophan (W). (See also Creighton, Proteins, W. H. Freeman and Company(1984).) In addition, individual substitutions, deletions or additionsthat alter, add or delete a single amino acid or a small percentage ofamino acids in an encoded sequence are also “conservativesubstitutions.” Insertions or deletions are typically in the range ofabout 1 to 5 amino acids.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow. Further, to the extent not alreadyindicated, it will be understood by those of ordinary skill in the artthat any one of the various embodiments herein described and illustratedmay be further modified to incorporate features shown in any of theother embodiments disclosed herein.

EXAMPLES

The following examples illustrate some embodiments and aspects of theinvention. It will be apparent to those skilled in the relevant art thatvarious modifications, additions, substitutions, and the like can beperformed without altering the spirit or scope of the invention, andsuch modifications and variations are encompassed within the scope ofthe invention as defined in the claims which follow. The followingexamples do not in any way limit the invention.

Example 1 Expression and Purification of Exemplary Engineered MBLMolecules

Engineered MBL for optimized binding to pathogens without the complementactivation and coagulation side effects which are present in WT MBL wereconstructed. The MBL carbohydrate recognition domain & various lengthsof the neck domain were cloned and fused to the Fc fragment of humanIgG1 comprising the hinge, CH2 and CH3 regions to form the fusionproteins. In one embodiment, the MBL carbohydrate recognition domain andat least a portion of the neck domain was cloned and fused to the Fcfragment of human IgG1 to form the fusion protein FcMBL.81 (SEQ ID NO.6). In one embodiment, the MBL carbohydrate recognition domain without aneck region was cloned and fused to the Fc fragment of human IgG1 toform the fusion protein FcMBL.111 (SEQ ID NO. 8). The complement andcoagulation activation regions of the MBL (e.g., the collagen triplehelix and hinge MASP binding regions) was removed from the fusionproteins.

In some embodiments, the AKT tripeptide was inserted into the N terminusof Fc (at the hinge region: H of the Fc-X vector shown in FIG. 3) forsingle-site biotinylation of the FcMBL.81 (The amino acid sequence forsuch embodiment with the AKT tripeptide fused to the N terminal portionof the Fc, designated as AKTFcMBL.81, is shown in SEQ ID NO. 7). Themono-biotin engineered MBL molecules AKTFcMBL.81 were then conjugated tostreptavidin-coated beads and the carbohydrate binding MBL heads wereoriented away from the substrate for optimized binding to pathogens.

In some embodiments, the asparagine N82 (N297 in Kabat numbering) of SEQID NO. 6 was mutated to aspartic acid (D) to remove the glycosylation ofFc to remove antibody dependent cellular cytotoxicity (ADCC) andComplement Dependent Cytotoxicity (CDC) functionality.

Four different engineered MBL fusion protein construct were produced:

-   (1) Fc MBL.111 (SEQ ID NO. 8) consists of the Fc portion of IgG    (Kabat numbering 216-447) fused to the MBL CRD head (amino acids 111    to 228).-   (2) Fc MBL.81 (SEQ ID NO. 6) consists of the Fc portion of IgG    (Kabat numbering 216-447) fused to the MBL CRD head AND neck region    (amino acids 81-228).-   (3) AKT-Fc MBL.81 (SEQ ID NO. 7) consists of the Fc portion of IgG    (Kabat numbering 216-447) fused to the MBL CRD head AND neck region    (amino acids 81-228). The 3 amino acid fragment AKT is fused to the    N terminal portion of the Fc.-   (4) Fc MBL.81 D consists of the Fc portion of IgG (Kabat numbering    216-447) fused to the MBL CRD head AND neck region (amino acids    81-228), in addition, the Fc glycosylation site has been removed by    substituting aspartic acid (D) for asparagine (N) at position 82    (297 in Kabat numbering)

A major advantage of the Fc fusion technology is the ease of expressionand purification of fusion proteins (Lo et al. (1998) ProteinEngineering. 11: 495-500). The N terminal Fc has been shown to improveexpression levels, protein folding and secretion of the fusion partner.In addition, the Fc has a staphylococcal protein A binding site, whichis extremely useful for one-step purification on protein A affinitychromatography. Thus, in some embodiments, different engineered MBLnucleic acid sequences encoding the amino acid sequences discussed abovecan be inserted in the Fc-X vector disclosed in the Lo et al. Id. HumanU 293 cells were then transfected with Fc MBL DNA using thelipofectamine reagent (Invitrogen). The engineered MBL fusion proteinscan be purified on a 5 ml HiTrap Protein A column using the GE AktaAvant 25 system.

For protein purification, an exemplary loading buffer is 100 mMPhosphate 150 mM NaCl pH 7, and an exemplary elution buffer is 100 mMPhosphate 150 mM NaCl pH 3. Following elution, the protein wasimmediately neutralized with 1 N NaOH and Tween 80 (Pierce SurfactAmp)was added to a final concentration of 0.01%. The engineered MBL fusionproteins were then sterile-filtered through a 0.22 micron nylon filterand stored at 4° C. This one-step purification gave a recovery of atleast about 90% (data not shown).

To analyze the purified proteins, a reduced SDS-PAGE was performed usingthe Invitrogen System. Western blotting was then performed onto PVDFmembranes using an iBlot system (Invitrogen) and the PVDF membranes werethen probed with biotinylated anti-human MBL antibodies (R&D Systems).The results of the purified proteins FcMBL.81 and MBL wild-type (WT) areshown in FIG. 4.

Example 2 Testing the Potency/Biological Activity of the MBL Constructs

To determine calcium-dependent binding of the Fc MBL proteins to amannan-coated ELISA plate, 96-well ELISA plate was first coated with 0.5mg/ml mannan (M3640, Sigma). The purified Fc MBL.81 and Fc MBL.111fusion proteins (supernatant from 293 cell expression purified usingrecombinant protein A using the AKTA system & confirmed >90% pure bySDS-PAGE) were diluted and added to the mannan-coated ELISA plate. Insome sample wells, EDTA was also added to chelate calcium. A secondaryantibody anti-human Fc HRP (109-036-098 Jackson Lab) was then added toall sample wells. The O.D. values of each sample were measured at 450nm.

Presented herein indicated that the Fc MBL.81 fusion protein binds tomannan in the presence of calcium, but such binding is reduced by ˜100fold in the presence of EDTA (FIG. 5). These assays can be repeated andcompared with the WT MBL from SinoBiologicals. As shown in FIG. 5, theFcMBL.111 fusion protein is inactive in the mannan binding, regardlessof the presence of calcium. Without wishing to be bound by theory, theneck regions are needed in some embodiments provided herein to provideflexibility and orientation of the engineered microbe-targetingmolecules (e.g., engineered MBL molecules) for binding to thecarbohydrates on pathogens. The findings presented herein indicate thatboth the Fc MBL.81 and WT MBL binding to mannan is calcium-dependent andcan be reversed by EDTA chelation. Further, the findings indicated thatFc MBL.111 (the MBL CRD head fused to Fc) appears to be a relativelypoor binder to mannan, as compared to Fc MBL.81.

The AKT-FcMBL.81 fusion protein appears to show a higher backgroundbinding to mannan in the presence of EDTA than the Fc MBL.81 fusionprotein. This can be, not being bound by theory, due to the AKT bindingsite on the N terminus of the fusion protein, which is designed foraminoxy biotinylation for oriented binding on streptavidin beads. Thus,the AKT-FcMBL.81 fusion protein can be a bit sticky. In someembodiments, the AKT-FcMBL.81 may not be ideal for the mannan bindingassay.

Example 3 Activity in Complement and Coagulation Assays with MBL NullSerum

WT MBL activates complement and coagulation through the MASP proteins.In this example provided herein, MBL null serum was used as a source ofcomplement and coagulation proteins, while the WT MBL and the FcMBL.81were used as the sources of MBL to activate complement activation ofclotting function.

Assays to measure complement activation has been discussed in Michelowet al. (2010) JBC 285: 24729. Briefly, triplicate samples of dilutedchimeric proteins were added to mannan-coated microtiter plates with 1%MBL-null human serum as a source of MASP. Normal human serum complementstandard (Quidel, San Diego, Calif.) containing native MBL was used togenerate a standard curve. After incubation at 37° C. and rinsing,deposited human C4 fragments (Sigma-Aldrich) were detected withanti-human C4c antibodies (Dako Denmark A/S), followed by addition ofbiotinylated secondary antibodies (JacksonImmunoResearch Laboratories,West Grove, Pa.), avidin-containing Vectastain ABC-alkaline phosphatasereagent (VectorLaboratories, Burlingame, Calif.), and p-nitrophenylphosphate, and measurement at A405 nm.

Methods to determine coagulation and search for thrombin-like activityhave been previously established, e.g., discussed in Takahashi et al.(2011) Immunobiology 216: 96). Briefly, the assay was designed to detectMBL-MASP complex-mediated activities by using plates that were coatedwith Mannan in carbonate binding buffer, pH 9.5. After rinsing theprepared mannan-coated plates with TBS, pH 7.4, supplemented with 10mMCaCl₂ (TBS-CaCl₂), the wells were incubated with diluted MBL proteinswith or without 1% MBL null serum as a MASP source. The wells wereincubated at room temperature for 1 h and then rinsed thoroughly to washoff endogenous prothrombin and thrombin. Thrombin-like activity ofMBL/MASP complex was measured by incubating the wells with a rhodamine110 based thrombin substrate (tosyl Gly-Phe-Arg-amide, R22124,Invitrogen).

The findings presented herein indicate that no C4 deposition onmannan-coated plates from Fc MBL.81 activation of MBL null serum, but C4was deposited from the WT MBL control (Data not shown). Further, nocoagulation activation (thrombin-like activity) by the Fc MBL.81 fusionprotein was determined (data not shown). Thus, unlike the WT MBL, the FcMBL.81 does not activate complement or coagulation.

Example 4 Exemplary Methods for Production of MBL Magnetic Microbeads

Different microbe-targeting molecules (e.g., engineered MBL molecules)can be coupled to magnetic microbeads using different sizes or types ofmicrobeads and surface chemistries. Examples of magnetic microbeads thatcan be used for the microbe-targeting magnetic microbeads include, butare not limited to, 1 micron MYONE™ T1 streptavidin microbeads(streptavidin coupled via tosyl groups) from Invitrogen (DYNABEADS®), 1micron Tosylactivated microbeads from Invitrogen (DYNABEADS®), and 100nm (128 nm average diameter) Streptavidin Plus microbeads (streptavidincoupled via carboxyl groups) from Ademtech.

For binding to the MYONE™ and Ademtech streptavidin microbeads, the MBLcan be biotinylated (˜4 biotins per molecule) using Thermo EZ-LinkSulfo-NHS-LC-Biotin, which reacts with primary amines (lysine residues).For single-site biotinylation, for example, using the method describedin Witus et al. (2010) JACS. 132: 16812, the AKT-FcMBL.81 fusion proteinis biotinylated only at the N-terminal amine for oriented binding toStreptavidin microbeads. Briefly, the PLP-mediated bioconjugation is atwo-step process, in which an aldehyde is first added to the N-terminalamine in a PLP-mediated transamination reaction, followed by theaddition of aminooxy-biotin to the aldehyde.

For the Tosylactivated microbeads, the MBL can be directly andcovalently coupled to the surface of the microbeads by replacing thetosyl groups with its primary amines (lysine residues). Alternatively,aminooxy chemistries can be used to allow for oriented binding to theTosylactivated DYNABEADS® microbeads without using biotin-streptavidin.This should lead to a more stable system and reduce non-specific bindingto sticky streptavidin.

Example 5 Comparison of C. albicans Capture by Fc MBL.81-coated MagneticMicrobeads with WT MBL-coated Magnetic Microbeads

It was sought to determine whether the small ˜90 kDa AKT-FcMBL dimersassembled on the surface of the DYNABEADS® microbeads with the CRD headsoriented away from the microbead substrate, and whether the AKT-FcMBL.81conjugated to the magnetic microbeads (via biotin-streptavidin coupling)has the same avidity of binding as the large (˜650 kDa) multimericwild-type biotinylated MBL, which is randomly attached to thestreptavidin microbead.

Briefly, the 650 kDa MBL from Sino Biological was biotinylated and thencoupled to MYONE™ Streptavidin microbeads described in Example 4. Thewide-type MBL coupled to such microbeads was designated as “Sino MBLmicrobeads” below. AKT-FcMBL.81 was also coupled to MYONE™ Streptavidinmicrobeads. Equivalent masses of the two proteins were coupled to equalnumbers of microbeads.

To perform the MBL microbead capture experiments, 1500 Candida wereincubated for 10 minutes with either 1 ul of the AKT FcMBL microbeads or1 ul of the Sino MBL microbeads in TBS-Tween buffer supplemented with 5mM Calcium. Microbeads with bound Candida were removed by capturing themon a magnet for 2 minutes. Then, about 1/10 of the captured material wasplated on YEPD Agar plates and incubated at 30° C. for 1-2 days,followed by counting and comparing with the total counts from anequivalent dilution of the 1500 Candida starting culture.

FIG. 6A shows that greater than 95% of the Candida was bound to the AKTFc MBL.81 microbeads, while greater than 92% was bound to the Sino MBLmicrobeads. There were no significant differences in the results usingthese two types of microbeads, indicating that the engineered MBLmagnetic microbeads described herein showed at least comparable, orindeed better, binding than the WT MBL magnetic microbeads at theindicated pathogen density.

Next, it was sought to evaluate the performance of the engineered MBLmagnetic microbeads at higher pathogen densities, e.g., above 10⁸ yeastcells. First, C. albicans was cultured overnight for 2 days, and thenwashed 2 times with PBS. The final pellet of C. albicans was resuspendedin T-TBS w/ Calcium [TBS, 0.1% Tween-20, 5 mM CaCl₂] with a reading ofOD600 around 0.5-0.7. The unlabeled MYONE™ T1 streptavidin microbeadswere washed 2 times in PBS 0.1% BSA and diluted in original volume ofPBS 0.1% BSA. To a 1.5 mL tube, about 1 mL of above C. albicans mixturewas first added, followed by either 2 μl of unlabeled microbeads, 2 μlof FcMBL labeled microbeads, or 2 ul of WT MBL labeled microbeads. A C.albicans mixture was used as a no-bead control. All the mixtures werethen mixed on a Hula mixer for about 10 minutes, followed by capturingthe microbeads on a magnet for 2 minutes. The OD of unbound fraction(supernatant) was measured at 600 nm.

Alternatively, Candida were cultured until the OD600 reached a value of−0.6. The yeast were incubated for 10 minutes with either 1 ul of theAKT FcMBL microbeads or 1 ul of the WT MBL microbeads in TBS-Tweenbuffer supplemented with 5 mM Calcium. The microbeads and captured yeastwere removed by magnetic capture for 2 minutes. The OD (600 nm) of theremaining supernatant was measured to determine the unbound fraction.

The oriented AKT-FcMBL.81 microbeads demonstrated significantly betterbinding performance than (sino) WT MBL microbeads in pull-down bindingassays using the MYONE™ Streptavidin 1 micron beads (FIG. 6B). Thus, thefinding indicates that the AKT-FcMBL.81 microbeads have a higher bindingcapacity than WT MBL microbeads.

Example 6 Effect of Magnetic Microbead Sizes on Efficiency of PathogenCapture

To determine the optimal microbead size for capturing both fungi andbacteria, the binding of Candida to microbeads (with a size rangingbetween ˜1 μm and ˜128 nm) coated with AKT Fc MBL was evaluated usingthe Pull-down assay described in Example 5. Surprisingly, the captureefficiency increases with decreasing sizes of magnetic microbeads. Thisreverses the hypothesis that the larger microbeads were better for fungiand that the ˜100 nm were better for bacteria but would be sub-optimalfor fungi. Thus, in some embodiments, the ˜100 nm-microbeads (e.g., ˜128nm) can be used for both bacteria and fungal capture.

Example 7 Binding Performance of Engineered MBL Magnetic Microbeads inSaturated Vs Log Phase Growth Candida Cultures

As shown in Example 5, static cultures of Candida were bound strongly bythe engineered MBL microbeads. Next, it was sought to determine if therewas any difference in microbead performance using Candida in log-phasegrowth.

First, C. albicans 2-day old overnight culture and log-phase culture(OD600=˜0.5) were washed 2 times with PBS. The final pellet of C.albicans was resuspended in T-TBS w/ Calcium [TBS, 0.1% Tween-20, 5 mMCaCl₂] with a reading of OD600 around 0.3-0.5. The unlabeled MYONE™ T1streptavidin microbeads were washed 2 times in PBS 0.1% BSA and dilutedin original volume of PBS 0.1% BSA. To a 1.5 mL tube, about 1 mL ofabove C. albicans mixture was first added, followed by either 2 μl ofunlabeled microbeads, 2 μl of FcMBL labeled microbeads, or 2 μl of WTMBL labeled microbeads. A C. albicans mixture was used as a no-beadcontrol. All the mixtures were then mixed on a Hula mixer for about 10minutes, followed by capturing the microbeads on a magnet for 2 minutes.The OD of unbound fraction (supernatant) was measured at 600 nm.

FIG. 8 indicates that there may be a difference in AKTFcMBL and WT MBLcapacity to pull down C. albicans depending on the growth state of theyeast, but the variance in the log phase growth is relatively noisy. Oneof skill in the art can determine the pathogen capture efficiency ofdifferent embodiments of engineered microbe-targeting magneticmicrobeads described herein with varying pathogen densities, e.g., usingthe methods described herein.

Example 8 Evaluation of the Engineered MBL Magnetic MicrobeadPerformance in Human Blood Samples

Blood samples containing bacteria and bacterial debris can beefficiently cleansed/captured using engineered MBL magnetic microbeads.The amount of bacteria and bacterial debris present in blood can bereliably determined using the FcMBL ELISA (see FIG. 10, Example 9). Theblood samples spiked with either E. coli or S. aureus were complementedwith 5 mM calcium (final concentration) and with 4 mg/ml heparinfollowed by binding/clearing with the engineered MBL magneticmicrobeads. The performance of the engineered MBL magnetic microbeads inspike-in blood was assessed by FcMBL ELISA as described later.

The clearing of bacteria/bacterial fragments from a blood sampleimproved when iterative captures were performed, e.g., due to saturationof the FcMBL engineered beads in the first binding run.

Example 9 Colorimetric ELISA for Detecting Pathogen

Presented herein is a colorimetric ELISA (Enzyme linked ImmunosorbentAssay) kit developed for detecting pathogens, which can integrate intothe existing workflow and capabilities of a typical laboratory (e.g., apathology laboratory). In some embodiments, the ELISA kit can compriseengineered microbe-targeting magnetic microbeads (e.g., Fc MBL or AKTFcMBL-coated magnetic microbeads), HRP-labeled Fc MBL or AKTFc MBL. Othersecondary reagents (e.g. HRP (Horse Radish Peroxidase) labeledantibodies) can also be included in the ELISA kit described herein. TheHRP labeled proteins & antibodies can diffuse into the clumps ofpathogen and magnetic microbeads. The HRP enzyme can amplify thedetection signal, thus improving the sensitivity of the assay. SuchELISA kit can be used with typical laboratory experiments that work withmagnetic microbeads in a microtiter plate, e.g., KingFisher 96-wellmagnetic bead washer.

In some embodiments of the ELISA assays described herein, themicrobe-targeting magnetic microbeads, e.g., FcMBL magnetic microbeads,can capture microbes or pathogens in a sample, followed by detectionwith microbe-targeting HRP (e.g., FcMBL-HRP or Wheat Germ Agglutinin(WGA)-HRP). Such assays can be used to determine the presence of anunidentified pathogen. A schematic diagram showing one or moreembodiments of the ELISA assays comprising microbe-targeting magneticmicrobeads is shown in FIG. 10.

In other embodiments of the ELISA assays described herein, themicrobe-targeting magnetic microbeads, e.g., Fc MBL magnetic microbeads,can capture microbes or pathogens in a sample, followed by detectionwith specific antibodies depending on various pathogen tests. Forexample, anti-gram positive or anti-gram negative antibodies can be usedin a rapid Gram test, or specific anti-Salmonella antibodies can be usedin a typhoid test.

The ELISA assays provided herein can be automated, e.g., by employingthe existing capabilities of typical laboratory equipments, e.g., a96-well assay system coupled with a KINGFISHER™ magnetic bead washsystem.

An exemplary protocol for determining the limit of detection (LOD) ofsuch ELISA assay is described below:

-   a. Capture a microbe solution (e.g., E. coli dilutions) with    engineered microbe-targeting magnetic microbeads described herein    (e.g., FcMBL.81 magnetic microbeads) for about 15-minute incubation-   b. Isolate the magnetic microbeads on a magnet for about 2 minutes,    followed by four washes with a detergent, e.g., TBS-T 5 mM Ca²⁺-   c. Detect the bound microbes with a microbe-targeting HRP reagent,    e.g., FcMBL.81 HRP, reagent (for about 20 mins) in a blocking    buffer, e.g., a 6% BSA block-   d. Perform a detection assay with addition of a HRP substrate, e.g.,    TMB (3,3′,5,5′-tetramethylbenzidine) chromogen (incubation for about    5 min).-   e. Stop the reaction with an acid, e.g., 4 N H₂SO₄-   f. Measure O.D. at a certain wavelength depending on the enzyme    substrate used (e.g., 450 nm for TMB chromogen)

An exemplary graph of O.D. against varying concentrations of microbes orpathogens can be plotted for data analysis (FIG. 12). It should be clearthat any other ELISA protocols established in the art can be adaptedherein for use with the engineered microbe-targeting magneticmicrobeads.

Example 10 Exemplary Rapid Pathogen Detection Methods Based onColorimetic Assays

Sepsis or “blood poisoning” by bacterial and fungal infection produces18 million cases per year worldwide resulting in over 6 million deaths.A particularly vulnerable group is the newborn population in developingcountries. Of the approximately 3.6 million newborn deaths each year,worldwide infections are responsible for 30% of these deaths, of which15% are attributed to sepsis. Of the 3.6 million deaths, 98% of theseare present in developing countries where the medical facilities arelimited.

When physicians suspect that a patient is suffering from bacteremia theymust act quickly: since bacteria can divide very rapidly, every hourlost before a correct treatment is administered can make a crucialdifference in patient outcome (Garnacho-Montero et al 2006 Critical Care10:R111). Speed is especially important for neonates as up to 50% ofneonatal deaths occur in the first 24 hours. Consequently, physiciansmust quickly establish whether the patient indeed has bacteremia, and ifso, what antibiotics to prescribe. The current gold standard foridentification of infection is blood culture, which generally takes daysand fails to identify a causative agent in more than 50% of cases.Therefore, there is a strong need, e.g., in developing countries, for apoint-of-care diagnostic assay/device that is portable, requires noelectricity, is easily read, is low cost, and/or is rapid.

Further, in studies from developing countries the majority of bloodstream infections have been caused by Staphylococci, Klebsiella andAcinetobacter, which together comprise more than 85% of the pathogensisolated. In studies in Boston, the major pathogens are Staphylococci,Enterococci, Klebsiella, Escherichia and Pseudomonas, which make up morethan 85% of the pathogens isolated. Therefore, there is a need for arapid test that can detect and quantify bacteria or fungi infection ofbody fluids that are normally sterile and free of pathogens. Inaddition, it would be advantageous to be able to classify the microbe orpathogen into Gram-positive or Gram-negative microbe in order to choosethe correct broad spectrum treatment option speedily.

Pathogen Extraction and Concentration:

As presented herein, magnetic microbeads that are coated with engineeredmannose binding lectin (MBL) can be used for extraction and/orconcentration of pathogens or microbes from blood. MBL is aninnate-immune-system protein that can adhere to most blood-bornepathogens, thus enabling the magnetic microbeads suitably selective forextracting and purifying bacterial and fungal pathogens from largesamples of body fluids, e.g., blood, CSF, synovial fluid and urine. Someembodiments of the engineered microbe-targeting molecules, e.g.,engineered MBL (Fc linked to mannose binding region of MBL) can be1000-fold lower in cost of production and do not activatecomplement/coagulation. Other alternatives to MBL include, but are notlimited to, antibodies, and other lectins. In some embodiments,engineered MBL-coated magnetic microbeads can be used for capturing oneor more microbes and/or pathogens in a test sample.

Exemplary Colorimetric ELISA Assay for Detecting and QuantifyingInfection:

FIG. 10 shows an exemplary scheme of an ELISA method for detection andquantification of blood borne pathogens using one or more embodiments ofthe engineered microbe-targeting molecules or substrates (e.g.,FcMBL-coated magnetic microbeads). A patient sample is mixed withmagnetic microbeads coated with a suitable capture agent, e.g., 1 μm or128 nm magnetic microbeads coated with FcMBL molecules (includingAKT-FcMBL molecules), and a suitable buffer, e.g., Tris buffered salinewith 5 mM calcium ions and Tween 20 detergent. In some embodiments, asuitable buffer can be Tris-buffered saline containing Tween 20, butwithout 5 mM calcium ions. Following a suitable incubation and/or mixingperiod, e.g., about 10 minutes or about 20 minutes on a mixer (e.g., aHULAMIXER™ from Invitrogen), the FcMBL-coated magnetic microbeads withcaptured pathogens can be collected using a magnetic stand (Invitrogen)and washed in, e.g., Tris buffered saline with 5 mM calcium with orwithout Tween 20 detergent, to remove blood products. The capturedpathogens can be detected and quantified by any methods known in the artand/or described herein, e.g., using chromogenic reagents such asHorseradish Peroxidase (HRP)-labeled FcMBL (which can detect theinfection caused by any microbes, e.g., bacterial or fungal microbes) orspecific antibodies against Gram-Positive bacteria, e.g., anti-LTAantibodies, or against Gram-Negative bacteria, e.g. anti-LPS antibodies,or against Candida fungi, e.g., anti-Candida antibodies.

The level of infection or the amount of microbes captured onFcMBL-coated magnetic microbeads can be quantified, for example, bycomparing the test samples against standard curves of reference (e.g.,laboratory strains of bacteria or fungi) run in parallel. For example,FIGS. 20A-20B show data for capture efficiency of clinical isolatesassessed by FcMBL ELISA as described herein. Briefly, about 10 μg FcMBLmagnetic microbeads (˜1 μM) was added to about 10 μL of bacteria in thepresence of calcium ions (e.g., 1 mL TBST-Ca^(2±)). The capture wasagitated at about 900 rpm for about 10 mins at about 25° C., and ELISAwas performed on, e.g., THERMO-LABSYSTEM KINGFISHER™ Magnetic ParticleProcessor, using HRP-labeled FcMBL reagents.

An example of FcMBL-based ELISA detecting C. albicans captured fromblood is shown in FIG. 11, which shows that less than 500 Candida fungicells in blood can be detected using an embodiment of the FcMBL-basedELISA.

The sensitivity of the MBL sandwich ELISA for detecting E. coli insuitable buffers was evaluated and shown in FIG. 12. The limit ofdetection (LOD) for E. coli in one embodiment of the FcMBL-based ELISAassay was about or below 160 bacteria. Additionally, the captureefficiency of clinical isolates from different body fluids was assessedby FcMBL ELISA described herein (FIG. 21A). Briefly, about 10 μg FcMBLmagnetic microbeads (˜1 μM) was added to about 10 μL of bacteria spikedin a ˜1 mL to 2 mL mixture of fluid sample (e.g., blood, urine, CSF,sputum) and TBST-Ca²⁺ at a 1:1 volume ratio. The capture was agitated atabout 900 rpm for about 20 mins at about 25° C., and ELISA was performedon, e.g., THERMO-LABSYSTEM KINGFISHER™ Magnetic Particle Processor,using HRP-labeled FcMBL reagents. FIG. 21A shows that laboratory strainsand clinical methicillin-resistant S. aureus (MRSA) can be isolated fromblood, while N. Meningitidis appears to produce high background signalusing one embodiment of the FcMBL ELISA described herein. FIGS. 21A-21Bshows that S. aureus and E. coli spiked into different body fluids suchas blood, CSF, and urine can be detected using one or more embodimentsof the FcMBL ELISA described herein, while sputum appears to producehigh background signal using one embodiment of the FcMBL ELISA describedherein.

In some embodiments, the ELISA assay can comprise capture of a microbeor pathogen from blood with one or more embodiments of the FcMBL-coatedmagnetic microbeads (128 nm or 1 micron sized magnetic microbeads coatedwith one or more embodiments of FcMBL proteins) and detection eitherwith labeled-FcMBL (e.g., HRP-labeled FcMBL) for non-specific detectionof bacteria, or with labeled antibodies for specific detection of, e.g.,but not limited to, Gram-positive bacteria, Gram-negative bacteria, orfungi.

In one embodiment, the FcMBL-HRP or FcMBL-AP construct was generatedusing LIGHTNING-LINK™ HRP Conjugation Kit or LIGHTNING-LINK™ APConjugation Kit (Innova Biosciences), which is a lyophilized HRP or APmixture for directional coupling to antibodies and other proteins. Thecreation of FcMBL-HRP or FcMBL-AP can use any othercommercially-available kits as any labeling procedures for antibodieswell known in the art can be used.

Exemplary Manual Dipstick or ELISA Test:

Two exemplary forms of a rapid diagnostic assay, e.g., for a point ofcare diagnostic, were developed and assessed. These rapid diagnosticassays can be used in developing countries as they are portable, easilyread, low cost, rapid, and require no electricity. The exemplaryschematics of the two diagnostic assays are shown in FIGS. 13-14. FIG.13 shows an exemplary schematic of a manual dipstick assay for pathogendetection, and FIG. 14 shows an exemplary schematic of a manual ELISAtest for pathogen detection.

In one embodiment, the dipstick test requires capture of the pathogen toa membrane upon which the colorimetric readout is determined from. Theattachment of the FcMBL to the membrane can be performed with multipleapproaches, for example, by direct cross-linking FcMBL to the membrane,cross-linking FcMBL to the membrane via a nucleic acid matrix (e.g., DNAmatrix) for orientation and concentration (in a manner similar toFcMBL-coated magnetic microbeads), using FcMBL-coated magneticmicrobeads in combination with a focused magnetic field gradient appliedto the membrane, or any other art-recognized methods.

FIG. 15 shows results for a general dot blot detection of bacteria on amembrane. Serial dilutions of either E. coli or S. aureus were attacheddirectly to a Biodyne membrane, which was then blocked in 1% casein,incubated for 20 min with alkaline phosphatase (AP)-labeled FcMBL,washed, and detected colorimetrically with a BCIP/NBT reagent. Thesensitivity of the assay was about 200 cfu/ml to about 300 cfu/ml.

FIG. 16 shows results for a dot blot detection of bacteria on a membranecoupled with FcMBL. In one embodiment, a membrane (e.g., Biodynemembrane) is attached with FcMBL molecules at a certain concentration.Bacteria (e.g., S. aureus) was added to the FcMBL-Biodyne membranes,which were then blocked in 1% casein, incubated for 20 min with alkalinephosphatase (AP)-labeled FcMBL, washed, and detected colorimetricallywith a BCIP/NBT reagent. As shown in FIG. 16, the capture and detectionof the bacteria is FcMBL concentration dependent. As described earlier,in some embodiments, FcMBL can be directly immobilized on a membrane. Inother embodiments, FcMBL can be coupled to a membrane by a nucleic acidmatrix (e.g., DNA matrix). In alternative embodiments, FcMBL can becouple to any surface other than a membrane, e.g., a paper substrate,for the dipstick assay.

Any existing ELISA protocol can be used in combination with theengineered microbe-targeting molecules or substrates as described hereinfor microbe detection. Below shows an example of a protocol for anELISA-based microbe detection method carried out in a blood collectiontube (e.g., a modified blood VACUTAINER® optionally containing one ormore anti-coagulants such as citrate, phosphate, and dextrose (CPD) asshown in FIG. 14. The numeric steps below correspond to the numericvalues indicated in FIG. 14.

-   1. Add a test sample, e.g., blood, to one or more embodiments of the    microbe-targeting molecules or substrates (e.g., FcMBL-coated    magnetic microbeads). For example, about 10 μg of FcMBL magnetic    microbeads (e.g., at a concentration of about 2 mg/mL) can be added    to a test sample.-   2. Resuspend the microbe-targeting molecules substrates (e.g.,    FcMBL-coated magnetic microbeads) in the test sample, e.g., blood.-   3. Add TBST (e.g., Tris buffered saline (TBS) with 0.05% Tween 80)    containing Ca²⁺ at ˜5 mM (e.g., ˜5 mM CaCl₂)-   4. Incubate the mixture (optionally with gentle agitation) for about    10 mins to capture microbes-   5. Collect the microbe-targeting molecules or substrates. For    example, if the microbe-targeting molecules or substrates are    FcMBL-coated magnetic microbeads, the microbeads can be collected    with a magnet, e.g., placing a magnet around the tube.-   6. Add TBST wash until a desired level (e.g., a wash fill line)-   7. Collect the FcMBL-coated magnetic microbeads—remove wash—repeat    steps 5 and 6 at least two times-   8. Add resuspended FcMBL-HRP (e.g., resuspending FcMBL-HRP    lyophilized in about 6% BSA buffer in ddH2O) or other desired    detection agent to the collected FcMBL-coated magnetic microbeads    and incubate for about 10 mins-   9. Collect the FcMBL-coated magnetic microbeads-   10. Add TBST wash to a desired level (e.g., a wash fill line)-   11. Collect the FcMBL-coated magnetic microbeads—remove wash—repeat    steps 9 and 10-   12. Add a substrate suitable for the detection agent (e.g., a    chromogenic substrate such as TMB for HRP-based detection) and allow    the reaction to develop for about 10 mins-   13. Collect the FcMBL-coated magnetic microbeads, e.g., with a    magnet.-   14. Transfer the reaction solution to a readout tube and compare the    color of the reaction solution to a reference (e.g., a reference    strip).

The reagents and steps as shown above are illustrated as an example andare not meant to be limiting. Thus, appropriate modifications toreagents and/or steps by a person having ordinary skill in the art arealso within the scope described herein. For example, different washbuffers, detection agents, and/or chromogenic substrates can be used.The number of wash steps can be increased or decreased, depending on thevolume of wash buffer used and/or incubation time. Some reagents (e.g.,FcMBL magnetic microbeads) for the assay can be supplied as lyophilizedand/or in sterile bottles. The readout of the assay can be based uponcomparison to a reference (e.g., a laminated color strip). In oneembodiment, the total assay time of the assay is approximately 1 hour.

In contrast to blood culture, some embodiments of the pathogen detectionassays or diagnostic assays described herein can detect bacteria and/orfungi in short times, e.g., as little as 1 hour. Further, additionaladvantages of some embodiments of the diagnostic assays (e.g.,point-of-care dipstick and ELISA assays as shown in FIGS. 13-14) caninclude, e.g.,

-   -   Portable: half of neonatal deaths occur in home childbirth        settings;    -   No electricity needed: manual operation;    -   Easy to read: colorimetric readout;    -   Low cost: no expensive instrument needed to read result;    -   Easy disposal: incinerate biohazard waste; or    -   any combinations thereof.        Accordingly, some embodiments of the pathogen detection assays        or diagnostic assays described herein can enhance        clinically-based diagnosis in regions without lab access, thus        reducing inappropriate use of antibiotics. Further, some aspects        described herein can reduce patient loss to follow-up by        enabling the diagnostic test and treatment        administration/prescription in same encounter. Additionally,        some aspects described herein can reduce exposure of neonates to        clinical setting.

Example 11 Regeneration of Engineered MBL Molecules (FcMBL) Using SodiumPhosphate Buffers and/or Acidic Buffers

FcMBL described herein can be used to capture a wide range of microbesfrom environmental and biological samples. In situations wherecontinuous cleaning or monitoring is required it would be useful to beable to use the same substrate (e.g., microbeads) throughout theprocess. This would require releasing captured microbes so the substrate(e.g., microbeads) could be reused. However, releasing captured microbesfrom the FcMBL microbeads can be difficult. While the initial binding ofFcMBL microbeads to microbes is calcium-dependent, after the microbesare bound, transferring the microbe/microbead mixture to a solutionlacking calcium generally does not lead to the release of the capturedmicrobe—presumably because of the high avidity between the microbeadsand microbes makes FcMBLs affinity for calcium too high to overcome bysimple dilution in a reasonable amount of time. Therefore, mechanismsfor actively removing the calcium from the FcMBL-microbe interactionwere evaluated herein.

The most common strategy used in the art for removing calcium is the useof calcium chelating agents (e.g. EDTA or EGTA). Unfortunately,chelating agents such as EDTA and EGTA can be harsh or dangerous tobiological samples, so additional mechanisms to actively remove calciumwere investigated. Two alternative strategies that were evaluatedincludes (i) the use of low pH buffers (acids) that can protonate thenegatively charged carboxyl groups (glutamate side chains) on FcMBL thatare responsible for binding calcium (protonating these side chains canremove their negative charge, which can remove their ability to bind topositively charged calcium ions) and (ii) the use of buffers in thatcalcium is not soluble such that the introduction of such buffers canlead to the precipitation of the calcium ions, making them unavailablefor the necessary interaction with the FcMBL-microbe interface.Specifically, 0.2M glycine buffer at pH 2.8 and 0.1M Sodium PhosphateBuffer at pH 6.0 (the solubility of calcium in phosphate buffer isextremely low) have been used and compared to 0.1M EDTA in Tris BufferedSalt. These conditions have been assessed on FcMBL microbeads bound tothe bacteria, E. coli, using an FcMBL-based ELISA.

Three different dilutions of an E. coli overnight culture were capturedon FcMBL microbeads, washed with one of four elution buffers including aTBST control, EDTA, 0.2M glycine (pH 2.8) and 0.1M sodium phosphate (pH6.0), and then run through a standard ELISA protocol. A decrease insignal corresponds to less E. coli bound to the FcMBL microbeads priorto the ELISA assau. As seen in FIGS. 18A and 18B, in addition to EDTA,both the low pH buffer (e.g., 0.2M glycine, pH 2.8) and the sodiumphosphate buffer were able to release bound E. coli from the FcMBLmicrobeads. Amount of released microbe can be increased by increasingthe incubation time or by combining the phosphate and acid conditions(i.e. phosphate buffer at a low pH).

Example 12 Use of the FcMBL as an Antibiotic or Antiseptic

S. aureus is the major cause of sepsis in wounds, burns and orthopedicsurgery. To determine whether the binding of S. aureus to FcMBLmicrobeads reduced the number and growth of bacterial colonies on agarplate culture, two equal aliquots of a 10⁻⁴ dilution of S. aureus wereplated onto identical LB agar plates and cultured overnight. One of thealiquots was mixed with FcMBL microbeads, and the mixture of microbeadsand pathogens was plated. The control aliquot was plated without anyFcMBL microbeads.

As shown in FIG. 19, the plate with the S. aureus mixed with FcMBLmicrobeads grew 220 colonies whereas the control grew more than 1000colonies.

Binding of the FcMBL microbeads with S. aureus reduces the number ofcolonies on overnight plating ˜5-fold, indicating that a wound dressingattached with FcMBL microbeads can enable the binding of S. aureus toFcMBL microbeads, thus reducing and localizing pathogen load. As such,the movement of the S. aureus deeper into the wound can be reduced.Localized pathogens attached to dressings can be easily removed duringregular dressing changes. In other embodiments, the FcMBL localizationtreatment can be combined with other wound dressing protocols e.g., butnot limited to, silver nanoparticles, negative pressure treatment,vacuum-assisted debridement. In alternative embodiments, FcMBLmicrobeads can be used to debride a fluid.

In some embodiments, FcMBL molecules are assessed as a therapeutic in ananimal model of sepsis, including, e.g., MBL knockout mouse model (See,e.g., U.S. Pat. No. 7,491,868, the content of which is incorporatedherein by reference), S. aureus model, and/or the rat sepsis model (See,e.g., Onderdonk A B et al., (1984) Rev Infect Dis; 6 Suppl 1:S91-5). Asurrogate molecule with mouse Fc g2a and human MBL as the human IgG1Fcare made immunogenic in mice. Fully mouse versions with mouse MBL-A and-C which work in both mice and rats are constructed.

Example 13 Elution of Bacteria Bound to FcMBL Molecules with VariousChelation, pH and Salt Buffers

A series of buffers with different chelating agents, pH, salt contentwere assessed in the 96 well ELISA assay to determine which buffer couldelute S. aureus or E coli off the FcMBL microbeads. An exemplary ELISAassay for detection of S. aureus or E. coli is described herein, and isnot construed to be limiting. Any other detection methods known in theart can be also used to detect readout signals of the target bacteria.

As described in Example 10, FIG. 10 shows the exemplary basis of theELISA assay using FcMBL-coated magnetic microbeads. The level ofinfection or the amount of microbes captured on FcMBL-coated magneticmicrobeads can be quantified by comparing the test samples againststandard curves, e.g., of laboratory strains of bacteria or fungi run inparallel. As shown in FIG. 23, using HRP-labeled FcMBL molecules as adetection agent in the ELISA assay can detect as few as 149 bacteria(e.g., S. aureus) in buffers. In some embodiments, the ELISA assay canbe performed in less than 1 hour.

The buffers that were assessed included, but were not limited to, 0.1Mphosphate buffer (pH 6); 0.1M phosphate buffer (pH 6) containing about150 mM NaCl; 0.1 M phosphate buffer (pH 6) containing 500 mM NaCl; 0.1 MEDTA in 0.1M phosphate buffer; 0.1 M EGTA in 0.1M phosphate buffer; 1Mborate buffer (pH 7.4); and 0.1 M carbonate-bicarbonate buffer. A bufferof TBST containing Ca²⁺ at a concentration greater than 1 mM was used asa control. Without wishing to be bound by theory, Ca²⁺ is generallyrequired for binding of microbes to MBL portion of the FcMBL molecule.After incubation for about 10-20 mins at room temperature (or up to 37°C.), elution of microbes bound to FcMBL magnetic microbeads wasanalyzed. As shown in FIG. 24, all the assessed buffers, except the onescontaining Ca²⁺, were able to elute greater than 85% of the E. colibound to the FcMBL molecules and/or magnetic microbeads, but they hadlittle or no effect on S. aureus. However, the borate buffer at 1M andpH 7.4 could elute about 33% of the S. aureus.

The elution of S. aureus and E. coli bacteria from FcMBL-coated magneticmicrobeads were also assessed using 0.1M EDTA or 0.1M phosphate buffer(pH 7.4) containing about 150 mM NaCl. The results are shown in FIG. 25Aand FIG. 25B as the OD450 and as a percent of bound bacteria,respectively. FIG. 25B shows that the EDTA and phosphate buffer canelute only 40% and 53% of the S. aureus off the FcMBL-coated magneticmicrobeads, whereas both the EDTA and phosphate buffer can removegreater than 90% of the E coli bacteria off the FcMBL-coated magneticmicrobeads and reduce the signal to about background level, indicatingthat the S. aureus can be bound more tightly to Fc portion of the FcMBLmolecules/magnetic microbeads than the gram-negative E. coli bacteria.

Example 14 Single Tube Assay for Detecting and/or Distinguishing S.aureus from E. Coli

Any existing ELISA protocol can be used in combination with themicrobe-targeting substrates as described herein for microbe detection.For example, an exemplary protocol for an ELISA-based microbe detectionmethod carried out in a modified blood collection tube (e.g., a modifiedblood VACUTAINER® optionally containing one or more anticoagulants suchas citrate, phosphate and dextrose (CPD)) is described earlier inExample 10 and shown in FIG. 14 and can be used to detect and/ordistinguish S. aureus from E. coli.

In some embodiments, the step 3 of the above-described exemplaryprotocol can employ TBST without calcium salts or calcium ions. In otherembodiments, the step 3 of the protocol can include a chelating agent(e.g., 50 mM EDTA or EGTA) in the TBST buffer with or without calciumions. In these embodiments, the absence of free calcium ions in the TBSTbuffer (e.g., either by addition of a chelating agent or absence ofcalcium ions into the TBST buffer) can reduce the likelihood of at leastE. coli, but not S. aureus substantially, binding to themicrobe-targeting substrates. Thus, S. aureus, but not E. coli, ispreferentially captured on the microbe-targeting substrates in theabsence of free calcium ions. In some embodiments, the step 3 of theabove-described exemplary protocol can employ TBST with calcium salts orcalcium ions, which allows at least both E. coli and S. aureus to becaptured on the microbe-targeting substrates.

In some embodiments, the washes involved in steps 6, 7, and 10 caninclude calcium salts (e.g., ˜5 mM CaCl₂) or calcium ions in the washbuffer, e.g., TBST. Thus, at least both E. coli and S. aureus can remainbinding to the microbe-targeting substrates. In other embodiments wherethe captured E. coli is desirable to be removed from themicrobe-targeting substrates, the washes involved in steps 6, 7, and 10can exclude calcium salts or calcium ions, and/or include a chelatingagent (e.g., ˜50 mM EDTA and EGTA) in the wash buffer.

As noted earlier, the reagents and steps as shown in Example 10 and FIG.14 are illustrated as an example and are not meant to be limiting. Thus,appropriate modifications to reagents and/or steps by a person havingordinary skill in the art are also within the scope described herein.For example, different wash buffers, detection agents, and/orchromogenic substrates can be used. The number of wash steps can beincreased or decreased, depending on the volume of wash buffer usedand/or incubation time. Some reagents (e.g., FcMBL magnetic microbeadsand/or FcMBL-HRP) for the assay can be supplied as lyophilized and/or insterile bottles. The readout of the assay can be based upon comparisonto a reference (e.g., a laminated color strip). In one embodiment, thetotal assay time of the assay is approximately 1 hour to 1.5 hours.

Using the exemplary ELISA assay protocol described above, FIGS. 26A-26Bshow the results of tube-based colorimetric ELISA assay for S. aureusand E. coli binding to FcMBL-coated magnetic microbeads in the presenceor absence of EDTA chelation. S. aureus and E. coli (10⁻¹ dilutionapproximately corresponding to about 10⁸ bacteria) were captured byFcMBL-coated magnetic microbeads in the presence or absence of calciumions and/or EDTA. For example, in some embodiments, after resuspensionof the FcMBL-coated magnetic microbeads in a test sample, e.g., blood, aTBST buffer (e.g., Tris buffered saline (TBS) with 0.05% Tween) withcalcium salts (e.g., ˜5 mM CaCl₂) can be added. In such embodiments,both E. coli and S. aureus can be captured on the FcMBL-coated magneticmicrobeads in the presence of calcium ions. In order to remove thecaptured E. coli from the FcMBL-coated magnetic microbeads, theFcMBL-coated magnetic microbeads with bacteria can be washed with TBSTwithout sufficient free calcium ions (e.g., TBST without a calcium salt,e.g., CaCl₂; a solution of a calcium salt (e.g., ˜5 mM CaCl₂) withexcess EDTA (e.g., ˜50 mM EDTA); or a EDTA solution (e.g., ˜50 mMEDTA)). In alternative embodiments, E. coli can be prevented frombinding to the FcMBL-coated magnetic microbeads when a test sample is incontact with FcMBL-coated magnetic microbeads, e.g., by using TBSTwithout sufficient free calcium ions to enable E. coli binding to theFcMBL-coated magnetic microbeads.

After microbe capture and washes, any remaining bacteria bound on theFcMBL-coated magnetic microbeads were then detected, e.g., by FcMBL-HRPand TMB colorimetric detection. The total assay time was about 40minutes. FIGS. 26A-26B show that unlike E. coli, S. aureus can bind tothe FcMBL in the presence of a chelating agent, e.g., EDTA, indicatingthat other than MBL-mediated binding, Fc-mediated binding can beinvolved. However, there can be additive binding of S. aureus to theFcMBL in the presence of calcium ions, as the binding of S. aureus tothe FcMBL in the presence of calcium ions is almost twice as strong asthat in the absence of calcium ions. This indicates that both the Fcbinding and the MBL binding can be responsible for the stable bindingbetween FcMBL and S. aureus. The kinetics of binding between FcMBL andS. aureus can be determined, e.g., on a BiaCore system, or KinExA.

It was next sought to determine if capture of S. aureus in the presenceof a chelating agent, e.g., EDTA, is selective. Accordingly, capture offour pathogenic species, e.g., E. coli, S. aureus, S. epidermidis and C.albicans, were compared in the presence or absence of a chelating agent,e.g., EDTA, and variable Ca²⁺ concentrations. FIG. 27 shows that S.aureus can be captured by FcMBL-coated magnetic microbeads in thepresence of a chelating agent, e.g., EDTA, while the other pathogenicspecies, e.g., E. coli, S. epidermidis and C. albicans requires calciumions for binding to the FcMBL-coated magnetic microbeads. In someembodiments, replacement of Ca²⁺ at high concentrations appears toreduce S. aureus capture on the FcMBL-coated magnetic microbeads. It isnoted that S. epidermidis, unlike S. aureus, requires calcium ions forbinding to the FcMBL-coated magnetic microbeads. Thus, capture and/orwash in the presence of a chelating agent, e.g., EDTA, can not only beused to distinguish S. aureus from E. coli, but can also be used todistinguish between S. aureus and S. epidermidis.

As S. aureus generally expresses protein A, which can contribute to theFc-mediated binding with the FcMBL, it was next sought to determine ifdisruption of Fc-mediated binding can cause S. aureus to elute off theFcMBL. Without wishing to be bound by theory, to disrupt Fc binding withprotein A, a low pH buffer can generally be used, e.g., pH 3 buffercontaining about 100 mM phosphate and 150 mM NaCl can be used; whereaschelation, e.g., using 50 mM EDTA, can generally be used to disruptMBL-mediated binding. However, as shown in FIG. 28, while E coli, asshown herein, can be eluted off the FcMBL-coated magnetic microbeadswith 50 mM EDTA pH 8, the S aureus is not significantly eluted by EDTApH 8 nor by a pH 3 buffer containing 0.1M Phosphate/0.15M Na⁺ pH 3 norby sequential washing with EDTA followed by the low pH phosphate buffer.As EDTA precipitates phosphate, the EDTA and low pH phosphate bufferwere not be able to be used together to determine if S. aureus could beeluted off FcMBL by disruption of both MBL-mediated and Fc-mediatedbinding. Nevertheless, the findings that S. aureus could not be elutedoff FcMBL by chelation or by reducing the pH indicate that there can beat least two independent mechanisms of binding the S. aureus to theFcMBL-coated magnetic microbeads. Without wishing to be bound by theory,chelation (which removes MBL—dependent binding) is not sufficient tocause S. aureus eluting off FcMBL because the Fc-dependent binding toStaphylococcal protein A is not affected and the low pH elution ofprotein A binding does not disrupt the MBL specific binding. (This canbe further assessed by using controls such as Fc-coated and wild-typeMBL-coated magnetic microbeads.) Accordingly, in some embodiments, it iscontemplated that concurrent disruption of both Fc-mediated andMBL-mediated binding between S. aureus and FcMBL can prevent S. aureusfrom binding to FcMBL. An exemplary low pH buffer that can work inconcert with EDTA chelation is 2M arginine at pH 4.4 (Arakawa et al.2004 Protein Expr Purif.; 36(2):244-2488). In one embodiment, 2 Marginine at pH 4.4 can be used to elute S. aureus off FcMBL and/orprevent S. aureus from binding to FcMBL.

The findings herein indicate that protein A present in the cell wall ofS. aureus can at least partly contribute to the ability of capturing S.aureus, rather than E. coli, in the presence of a chelating agent (e.g.,EDTA) due to the Fc-mediated binding. Thus, it is contemplated thatprotein A-expressing microbe can be captured on FcMBL in the presence ofa chelating agent (e.g., EDTA), and thus be distinguishable from proteinA-negative microbes, e.g., E. coli.

Example 15 Dot Blot/Dipstick Assays for Detecting and/or DistinguishingS. aureus from E. Coli

Dot blot and/or dipstick assays can be developed to capture microbe on asubstrate surface crosslinked with FcMBL upon which the colorimetricreadout is determined from. In some embodiments, the dot blot and/ordipstick assays can be used to distinguish S. aureus from E. coli.

The attachment of the FcMBL to the a substrate surface (e.g., membranesurface, glass surface, tubing surface) can be performed with multipleapproaches, for example, by direct cross-linking FcMBL to the substratesurface; cross-linking FcMBL to the substrate surface via a nucleic acidmatrix (e.g., DNA matrix or DNA/oligonucleotide origami structures) fororientation and concentration (in a manner similar to FcMBL-coatedmagnetic microbeads) to increase detection sensitivity; cross-linkingFcMBL to the substrate surface via a dendrimer-like structure (e.g.,PEG/Chitin-structure) to increase detection sensitivity; attractingFcMBL-coated magnetic microbeads to the substrate surface with a focusedmagnetic field gradient applied to the substrate surface, or any otherart-recognized methods. In some embodiments, the substrate surface canbe “oiled”. Without wishing to be bound by theory, the treating of asubstrate surface with an omniphobic layer can allow the binding to amicrobe by FcMBL without a subsequent hydrophobic binding between themicrobe and the substrate surface. This can allow chelation to removethe microbe when required. See, e.g., Wong T S et al., “Bioinspiredself-repairing slippery surfaces with pressure-stable omniphobicity.”(2011) Nature 477(7365): 443-447, and International Application No.:PCT/US 12/21928, the content of which is incorporated herein byreference, for methods to produce a slippery substrate surface. In someembodiments, the substrate surface can be further treated with ablocking agent (e.g., treatment with ˜1% casein for about 30 mins) toreduce any non-specific binding.

In some embodiments, the dipsticks attached with FcMBL can be added to atest sample, e.g., a blood sample, followed by one or more washes withTBST and incubation with alkaline phosphatase (AP)-labeled FcMBL (e.g.,˜20 mins of incubation with 1:10,000 dilution of AP-labeled FcMBL inTBST containing 3% BSA). After incubation with alkaline phosphatase, thedipsticks can be washed once or a plurality of times (e.g., at least 3washes with TBST followed by at least one wash with TBS) before additionof a BCIP/NBT reagent for colorimetric development (e.g., ˜20 mins). Insome embodiments, the wash buffers (e.g., TBST or TBS) can containcalcium ions or calcium salt (e.g., ˜5 mM CalCl₂) such that any microbeincluding E. coli and S. aureus can be captured on the dipsticks. Inalternative embodiments, the wash buffers (e.g., TBST or TBS) cancontain no calcium ions or calcium salts. In some embodiments, the washbuffers (e.g., TBST or TBS) containing calcium ions or calcium salt(e.g., ˜5 mM CaCl₂) can contain a chelating agent (e.g., ˜50 mM EDTA orEGTA) in excess to chelate free calcium ions. As shown herein, S. aureuscan remain bound onto FcMBL in the presence of a chelating agent or nocalcium ions. Accordingly, when the dipsticks after contact with a testsample, e.g., blood, are washed with buffers containing no free calciumions and/or a chelating agent, any bacteria on the dipsticks detectedafterward is likely S. aureus (as E. coli generally requires calciumions for MBL-mediated binding).

FIG. 15 shows results for a general dot blot/dipstick detection ofbacteria on a Biodyne membrane. Serial dilutions of either E. coli or S.aureus (10⁻¹ to 10⁻⁶ dilutions) were spotted directly onto a Biodynemembrane, which was then blocked in 1% casein, washed with TBSTcontaining ˜5 mM CaCl₂ once or at least two times, incubated for 20 minwith alkaline phosphatase (AP)-labeled FcMBL (1:10,000 dilution in TBSTcontaining 3% BSA and 5 mM CaCl₂, washed with TBST containing 5 mM CaCl₂at least three times followed by at least one wash with TBS containing 5mM CaCl₂, and detected colorimetrically with a BCIP/NBT reagent. FIG. 15shows that as low as 130 E. coli or 343 S. aureus can be detected after30-min development using AP-labeled FcMBL and BCIP/NBT detection system.To distinguish S. aureus from E. coli in a test sample, the dot blotsspotted with the test sample, e.g., blood, can be washed in the presenceof a chelating agent, e.g., EDTA. A microbe detected in the presence ofa chelating agent, e.g., EDTA, is likely S. aureus, rather than E. coli.

As described earlier, FIG. 16 shows results for a dot blot detection ofS. aureus bacteria on a membrane coupled with FcMBL. In one embodiment,a membrane (e.g., Biodyne membrane) is attached with FcMBL molecules ata certain concentration. Bacteria (e.g., S. aureus) was captured byFcMBL immobilized on the Biodyne membrane, which were then blocked in 1%casein (e.g., for about 30 mins), incubated for 20 min with alkalinephosphatase (AP)-labeled FcMBL, washed, and detected colorimetricallywith a BCIP/NBT reagent. As shown in FIG. 16, the capture and detectionof the bacteria is FcMBL concentration dependent. As described earlier,in some embodiments, FcMBL can be directly immobilized on a membrane.For example, about 1 μg to about 1 mg FcMBL, about 2 μg to about 500 μgFcMBL, about 5 μg to about 250 μg FcMBL, or about 10 μg to about 100 μgFcMBL can be spotted onto a Biodyne membrane and allowed to dry. In oneembodiment, the concentration of the FcMBL solution used for spotting onthe membrane can be about 0.1 mg/mL to about 25 mg/mL, about 0.5 mg/mLto about 20 mg/mL, about 5 mg/mL to about 15 mg/mL. In one embodiment,the concentration of the FcMBL solution used for spotting on themembrane can be about ˜11.5 mg/mL. In other embodiments, FcMBL can becoupled to a membrane by a nucleic acid matrix (e.g., DNA matrix). Inalternative embodiments, FcMBL can be coupled to any surface other thana membrane, e.g., a paper substrate, for the dipstick assay. In someembodiments, the substrate surface (e.g., Biodyne membrane) aftercoupling with FcMBL can be further treated with a blocking agent (e.g.,incubation with 1% casein for about 30 mins) to reduce any non-specificbinding. In some embodiments, the blocked substrate surface can bewashed with one or more washes, e.g., with TBST with or without calciumions (e.g., from a calcium salt such as CaCl₂). In some embodiments, theblocked substrate surface can be washed with at least two washes, e.g.,with TBST containing calcium ions (e.g., from a calcium salt such asCaCl₂).

An exemplary protocol for dot blot determination of E. coli and/or S.aureus is provided below:

-   -   Provide a Biodyne membrane spotted with about 1-100 μg (or about        3-15 μg) of FcMBL, which has been optionally blocked with about        1% casein for about 1 hour and washed at least two times in TBST        containing ˜5 mM Ca²⁺.    -   Dip the FcMBL-spotted membrane in a test sample, e.g., blood        sample    -   Add in TBST containing ˜5 mM calcium ions, and/or a chelating        agent (e.g., ˜100 mM EDTA), and incubate for about 20 mins to        allow bacteria captured by FcMBL. Addition of a chelating agent        (e.g., EDTA) can cause chelation of calcium ions, which can in        turn prevent/disrupt MBL-mediated binding, but not Fc-mediated        binding. In some embodiments, TBST containing ˜5 mM calcium ions        can be used to capture both E. coli and S. aureus, and E. coli        can then be eluted off with a TBST wash buffer containing a        chelating agent (e.g., EDTA).    -   Wash at least two times in TBST containing ˜5 mM calcium ions,        and/or ˜100 mM EDTA, and each wash can last for about 10 mins.        Addition of EDTA in the capture or wash buffer can cause        chelation of calcium ions, which can in turn prevent/disrupt        MBL-mediated binding, but not Fc-mediated binding. Thus, E. coli        cannot bind to FcMBL in the presence of a chelating agent, e.g.,        EDTA.    -   Optionally wash at least two times in TBST containing ˜5 mM        calcium ions.    -   Incubate, e.g., for about 30 mins, in alkaline phosphatase        (AP)-labeled FcMBL (e.g., 1:5000 dilution) diluted in TBST        containing about 3% BSA and −5 mM calcium ions    -   Wash at least three times with TBST containing ˜5 mM calcium        ions    -   Wash one or more times with TBS containing ˜5 mM calcium ions.    -   Develop with NBT/BCIP, e.g., for 4 min, for colorimetric        detection.

Any modifications to the exemplary protocol within one of skill in theart are also within the scope of different aspects and/or embodimentsdescribed herein. For examples, the number of washes can be increased ordecreased based on, e.g., the volume of a wash buffer used, how longeach wash takes, and/or binding affinity strength of bacteria to FcMBL.Further, different detection enzymes and corresponding enzymesubstrates, other than AP and NBT/BCIP, can be used, including, but notlimited to HRP and/or chromogenic substrates (e.g., TMB, DAB, and ABTS).In some embodiments, any chelating agent that can chelate calcium ions(e.g., EGTA, and EDTA) can be used. In some embodiments, any sources ofcalcium ions (e.g., different calcium salts such as calcium fluoride)that are compatible with the ELISA assay and binding of bacteria toFcMBL can also be used.

FIG. 29 shows that, using the exemplary protocol described above, S.aureus can be captured on FcMBL-spotted dot blots in the presence of achelating agent, e.g., EDTA, while E. coli cannot, because S. aureusexpress protein A, which can contribute to Fc-mediated binding, but E.coli do not. Thus, S. aureus can be distinguished from E. coli based onthe difference in binding behavior of S. aureus and E. coli to FcMBL inthe presence of a chelating agent, and in calcium ions.

Without wishing to be bound by theory, as protein G can generally bindto Fc of IgG, in some embodiments, the methods described herein can beused to detect protein G-expressing microbes (e.g., streptococci) anddistinguish them from protein G-negative microbes, e.g., E. coli.

Example 16 Rapid Identification of Microbes from FcMBL Bound MicrobialMatter or Component(s)

The diagnosis of infection relies on indirect or direct evidence. Theindirect evidence relies on the detection of an adapted and specifichost response directed against the pathogen. The direct evidence relieson the culture of the microorganism from the infected site,amplification and detection of pathogen-specific nucleic acids or thedetection of a specific antigen in blood or urine.

Specific antigen detection is widely used for a variety of infectiousdiseases, most commonly for legionellosis (Legionella pneumophilaserotype 1 in urine), malaria (Plasmodium falciparum in blood) and withless success with Streptococcus pneumonia infection (in urine). However,direct antigen detection can only be used to rule in or rule out aspecific etiology and cannot identify most bacteria.

As described herein, engineered microbe-binding molecules or substrates(e.g., FcMBL-bound paramagnetic microbeads) can be capable of bindingthe surface of a wide array of microbes including pathogens, e.g., butnot limited to, bacterial, fungal, parasitic or viral. For example, insome embodiments, blood or urine or any other biological fluid can besubjected to microbial capture by FcMBL coated magnetic microbeads andadequate controls (e.g., non-specific binding control by non-relevantprotein coated magnetic microbeads). Accordingly, engineeredmicrobe-binding molecules or substrates (e.g., FcMBL) can be used tobind microbes such as bacteria for diagnostic or therapeuticapplications.

Not only can the engineered microbe-binding molecules or substrates bindto at least a portion of a cell surface of a microbe, the engineeredmicrobe-binding molecules can also capture circulatingmicrobe-originating cell fragments or matter derived from microbes foundin biological fluids, e.g., in the course of an infection, even in theabsence of bacteremia. The presence of such elements can be used fordiagnostic applications, e.g., the presence of pathogen-originating cellfragments or matter derived from pathogens can be diagnostic of aninfectious disease. Moreover, the biochemical/proteomic (MALDI-TOF,multiple mass spectrometry (e.g., MSn) or specific antibody or aptamerbased) analysis of the bound products can allow the recognition ofelements pathognomonic for the most important pathogens. Accordingly,provided herein are also methods for diagnosis of infection occurring inany organ in the body of a subject (including blood, normally sterilefluids or virtual cavities) by capture of non-viable microbial matter orparticles circulating in blood, or found in other fluids such as urine,or in any other organ sampled by any appropriate means (e.g., but notlimited to, biopsy, puncture, aspiration, and lavage).

Binding of microbes or fragments thereof (including matter derived frommicrobes) can not only be used for infection of a sampled organ ortissue or cell(s) (blood or otherwise) but also to any major infectiousprocess ongoing anywhere in the body where sufficient bacterialdestruction or catabolism results in the presence of microbial matter inthe bloodstream, urine or any other conveniently accessed fluid.

The wide spectrum of FcMBL can enable the capture of most clinicallyrelevant bacterial species. As the presence of microbial matter orfragments of microbes can reflect deep tissue infection as theygenerally find its way into the bloodstream and most likely the urine,the capture and characterization of this microbial matter or fragmentsof microbes can be used as evidence markers specific for a givenmicrobial species, thus allowing the diagnosis and/or identification ofa microbe causing infection anywhere in an organism.

To this end, it was sought to determine if FcMBL could bind to microbialmatter including non-viable fragments or matter derived from a microbe,including endotoxin. The FcMBL-coated microbeads (e.g., FcMBL-coatedmagnetic or fluorescent microbeads) were incubated with bacterialcultures and later detected under a microscope. Specifically, theparamagnetic microbeads (1 μm diameter, MYONE™, Invitrogen) coated withFcMBL were used to capture E. coli and/or bacterial fragments thereofdiluted in TBST Ca²⁺5 mM for about 10 mins, followed by about 3 washes(the number of washes can be fewer or more, depending on the sampleprocessing conditions). The FcMBL-coated paramagnetic microbeads wereobserved under bright field. The captured E. coli and/or bacterialfragments could be also labeled with FcMBL-coated FluoroSpheres (e.g.,1:100 in TBST containing 5 mM Ca²⁺ and 3% BSA: incubation for about 2hours). All FcMBL binding matter was imaged using FcMBL coatedfluorospheres (Invitrogen). It was readily visible that both intactmicrobes and fragments thereof were captured by FcMBL-coated microbeads,as evidenced by observed outgrowth from bound intact microbes, ascompared to no outgrowth from bound fragments of a microbe (FIG. 30A).Further, the FcMBL-coated microbeads were incubated, e.g., for about 1hour, in the presence of Alamar Blue (AB) stain for detection of livecells and were imaged with an appropriate photo-excitation wavelength(e.g., SP5: yellow/green-FluoroSpheres; Red-AB). As shown in FIG. 30B,matter or material bound to FcMBL-coated fluorospheres and/or magneticmicrobeads can contain both live microbes (middle panel) and non-viablematter derived from microbes, e.g., E. coli (left panel).

The use of specific antibodies allows the characterization of the natureand/or nature of the microbial material bound to FcMBL. Without to belimiting, a specific antibody raised against Escherichia colilipopolysaccharide Lipid A (anti-LPS Lipid A antibody) or otherantibodies specific to a pathogen of interest was used in this Example.The E. coli was captured with 1 μm FcMBL microbeads as described herein,followed by incubation with a primary antibody specific to E. coli andoptionally a labeled secondary antibody that binds to the primaryantibody for imaging (if the primary antibody does not contain adetectable label). In one embodiment, the captured E. coli bound on theFcMBL microbeads was incubated with an anti-LPS lipid A antibody (e.g.,polyclonal antibody), for example, diluted by about 500-fold in TBSTcontaining Ca²⁺5 mM and 3% BSA for about 20 minutes, followed byincubation with an anti-goat IgG Cy3-labeled antibody, for example,diluted by about 2000-fold in TBST containing Ca²⁺5 mM and 3% BSA forabout 20 minutes. The labeled E. coli bound on FcMBL-coated microbeadswere then imaged by a fluorescent microscope. As shown in FIGS. 31A-31B,the E. coli-specific antibody (e.g., anti-LPS Lipid A antibody) wasshown to successfully bound to E. coli bound to FcMBL-coated substrates(e.g., magnetic microbeads or fluorescent microbeads). This binding wasobserved whether the capture of E. coli on FcMBL-coated magnetic beads(e.g., AKT-FcMBL-coated MYONE™ magnetic microbeads) was performed inbuffer or in blood with anti-coagulation agents such as heparin (FIG.31A) or EDTA (FIG. 31B). Microbeads incubated in blood or buffer withoutE. coli (e.g., not spiked with E. coli) were not found to be bound bythe anti-LPS Lipid A antibody. In addition, other antibodies (forexample anti-LTA antibodies) that are not reactive to E. coli strain didnot bind to the microbeads. Accordingly, characterization and/oridentification of microbes or fragments thereof bound onto engineeredmicrobe-binding molecules or substrates (e.g., FcMBL or FcMBL-coatedmicrobeads) can be achieved, e.g., by use of antibodies specific to themicrobe of interest.

In a rat sepsis model, samples (e.g., 200 μL) of blood and pleural fluidcollected from the animal after 24-hr infection were incubated with 1 μmFcMBL microbeads as described herein. In some embodiments, the blood wastreated with EDTA before the incubation with FcMBL microbeads.

In some embodiments, the FcMBL microbeads after incubation with abiological fluid sample (e.g., blood or pleural fluid) were furtherincubated with FcMBL-HRP for an ELISA assay as shown in FIG. 10. Theblood-EDTA sample collected from a rat after 24-hour infection producedan ELISA signal of OD450 nm at ˜0.8, while the pleural fluid samplecollected at the same time point produced an ELISA signal overflow.Similar trends were observed in results obtained from 72-hr samples.

In other embodiments, the FcMBL microbeads after incubation with abiological fluid sample (e.g., blood or pleural fluid) was furthersubjected to an antibody-based characterization as described above. Forexample, the captured microbes bound on the FcMBL microbeads wasincubated with an anti-LPS lipid A antibody (e.g., polyclonal antibody),for example, diluted by about 500-fold in TBST containing Ca²⁺5 mM and3% BSA for about 20 minutes, followed by incubation with an anti-goatIgG Cy3-labeled antibody, for example, diluted by about 2000-fold inTBST containing Ca²⁺5 mM and 3% BSA for about 20 minutes. The labeled E.coli bound on FcMBL-coated microbeads were then imaged by a fluorescentmicroscope. The samples from a rat sepsis model were characterized forthe presence of LPS on the FcMBL-coated microbeads (see FIGS. 32A-32B).The pleural effusion (ELISA OD—overflow) had widespread binding ofanti-LPS antibodies whereas the blood sample from the rat (ELISA OD—0.8)had some of defined signals (FIGS. 32A-32B), which can be representativeof intact E. coli.

When applied to the clinical samples that are positive by FcMBL ELISA,the specific detection of certain molecules (e.g., proteins,carbohydrates, lipids) present on a microbe surface such as Lipid A canallow further discrimination of positive samples or identification ofmicrobes present in the positive samples. In this regard, samples ofde-identified blood samples from a hospital were incubated with 1 μmFcMBL microbeads as described herein. The FcMBL microbeads afterincubation with the blood were first screened by further incubating withFcMBL-HRP for an ELISA assay as shown in FIG. 10. The FcMBL microbeadswere then further subjected to an antibody-based characterization asdescribed above. For example, in order to identify E. coli, the capturedmicrobes bound on the FcMBL microbeads was incubated with an anti-LPSlipid A antibody (e.g., polyclonal antibody), for example, diluted byabout 500-fold in TBST containing Ca²⁺5 mM and 3% BSA for about 20minutes, followed by incubation with an anti-goat IgG Cy3-labeledantibody, for example, diluted by about 2000-fold in TBST containingCa²⁺5 mM and 3% BSA for about 20 minutes. The labeled E. coli bound onFcMBL-coated microbeads were then imaged by a fluorescent microscope asshown in FIG. 33.

It was demonstrated herein that specific detection of LPS in FcMBLmicrobead bound microbes or microbial fragments was present in somepositive samples but none in FcMBL ELISA samples generating negative ornegligible signals (FIG. 33): this indicates that the use of a microbefamily-specific antibody allows the discrimination of the microbe fromwhich the captured material originates. For example, the sample (bottompanel) with a positive FcMBL ELISA signal did not demonstrate anybinding of anti-LPS antibodies to the FcMBL-coated microbeads,indicating that the microbes and/or microbial fragments bound to theFcMBL-coated microbeads were not associated with E. coli. (e.g., whenthe sample was infected with a gram-positive microbe). In contrast, thesample (middle panel) with a positive FcMBL ELISA signal demonstratedsubstantial binding of anti-LPS antibodies to the FcMBL-coatedmicrobeads, indicating that the microbes and/or microbial fragmentsbound to the FcMBL-coated microbeads were associated with E. coli or agram-negative microbe. Accordingly, such sample was determined to beinfected with E. coli and/or a gram-negative microbial infection. Moreimportantly, it should be noted that each of these de-identified sampleswere determined to be culture negative using traditional methods inpatients with clinical evidence of infection but no microbiologicaldocumentation. Accordingly, the use of engineered microbe-bindingmolecules or substrates (e.g., FcMBL or FcMBL-coated microbeads) is moresensitive and reliable than culture methods for diagnosis of aninfection.

The screening of a library of antibodies directed against the mostcommon microbes (including pathogens) can allow direct diagnosis ofmicrobe-specific infections anywhere in the body by a simple blood orurine test available in less than three hours in any microbiologylaboratory equipped for magnetic separation.

In a different embodiment, a rapid test could be performed using a“dipstick” format. For example, a membrane spotted with lines ofmicrobial species-specific antibodies (instead of FcMBL molecules asshown in FIG. 13) can be incubated with the FcMBL-coated microbeadspreviously incubated with the fluid tested. The FcMBL-coated microbeadscaptured by the proper antibodies can form a detectable band (e.g.,rust-colored for FcMBL-coated magnetic microbeads) on the membrane,indicating the species (one or many) of which microbial matter ormicrobes was captured.

Without wishing to be bound, while the Example demonstrates the use ofspecific antibodies to characterize and/or identify microbes present ina sample, other characterization methods such as mass spectrometriccharacterization methods can also be used. In some embodiments, theFcMBL microbeads with captured microbes and/or microbialmatter/fragments can be washed prior to any characterization methodssuch as mass spectrometric characterization methods.

In some embodiments, the FcMBL-coated microbeads with captured microbesand/or microbial matter/fragments can be subjected to direct MALDI-TOFanalysis for characterization and/or identification of species ofmicrobes and/or microbial matter bound to the FcMBL-coated microbeads.For example, the FcMBL-coated microbeads with captured microbialmaterials can be directly subjected to MALDI-TOF analysis.Alternatively, any art-recognized protocols can be applied on theFcMBL-coated microbeads to recover bound microbes and/or microbialcompounds/fragments prior to MALDI-TOF analysis. Exemplary methods torecover bound microbes and/or microbial compounds/fragments prior toMALDI-TOF analysis include, but are not limited to, Ca²⁺ chelation ofFcMBL-coated microbeads to release MBL bound material; lowering pH torelease Fc-bound protein A; protein extraction using formic acid andacetonitrile, and any combinations thereof. The control microbeads(e.g., non-FcMBL-coated microbeads) can be treated similarly forbaseline determination.

Extracted captured material from FcMBL-coated microbeads and/ornon-specific control-bound material can be subjected to massspectrometric analysis, including but not limited to, MALDI-TOF orMALDI-TOF-TOF. The non-specific control-bound material can establish abaseline for the composition of the medium tested. This profile can beused as reference for the analysis of the FcMBL-bound material. Peakspresent in the control-bound samples can be subtracted from the profileobtained from FcMBL-bound material.

The specific FcMBL bound material profile (e.g., after subtraction ofthe reference profile) can constitute the microbe signature. Bothpositive and/or negative charge analysis can be performed to identifyinformative peaks.

The microbe signature recognition can be analyzed by comparing thespecific FcMBL bound material profiles to microbe signature libraries,e.g., using algorithms based on the previously accumulated profiles suchas matching comparison algorithms.

For identification of microbe species, depending on origins of microbes,a microbe signature library can be established by in vivo or in situsamples such as clinical-trial derived samples and/or environmentderived samples (e.g., samples collected from a clinical setting,culture medium, food processing plant, water source). For example, blood(or other biological fluids) of patients infected with known microbes,e.g., pathogens, can be analyzed and a microbial material signature canbe characterized. Recognition of the signature in the same clinicalcontext can establish the family/genus/species diagnosis.

Additionally or alternatively, another microbe library can beestablished from in vitro analysis of FcMBL binding moieties of microbessubmitted to mechanical or chemical or antibiotic lysis or autolysis.The microbial material can be captured in different media, buffer,urine, blood or any appropriate medium.

The diagnostic profiles can be matched to any reference profiles, e.g.,specific in vivo or in situ derived microbe profiles and/or specificin-vitro derived microbes profiles for identification with a probabilityscore for generic infection, clades level, family level, genus level orspecies level identification.

Example 17 Performance Comparison of Colorimetric ELISA Using FcMBLMagnetic Microbeads and Conventional Blood Cultures

An animal model simulating intra-abdominal sepsis was produced byimplanting large bowel or cecal contents in the pelvic region of rats.The bowel or cecal contents were harvested from rats fed on a beef dietfor 2 weeks. Based on MALDI-TOF analysis, the cecal contents containeddifferent pathogens including Clostridium perfringiens, Enterobacteria,Enterococcus avium/raffinosus, and Enterococcus spp. Additional detailson creation of an animal model (e.g., a rat) with an intra-adominalsepsis can be found in Weinstein et al (1974) Infection and Immunity.10: 1250-1255 and Onderdonk et al. (1974) Infection and Immunity. 10:1256-1259.

In one experiment, the cecal contents (10⁹ bacterial cells) wereimplanted in the pelvic region of five rats. Rats were scarified atdifferent time points according to their morbidity after theimplantation and their morbidity ranking is shown in Table 1.

TABLE 1 Morbidity ranking of rats after implantation of cecal contentspelvic region of rats. Morbidity ranking Sacrifice time point (scale1-5) (hrs after implantation) Rat #1 1 (sickest) 10 hours Rat #2 2 18hours Rat #3 3 48 hours Rat #4 5 (the least sickest) 48 hours Rat #5 448 hours

The rats were sacrified and blood was collected for further analysis. Inorder to compare the performance of the conventional blood cultures andcolorimetric ELISA using FcMBL magnetic microbeads described herein(e.g., in Example 10), blood collected from the rats was analyzed by thetwo different methods. For conventional blood culture methods, the ratblood was cultured anaerobically for 4 days in different bacterialculture media (e.g., chocolate agar, sheep blood agar (SBA), Luria Broth(LB) and colistin Nalidixic Acid Agar (CNA) that is generally used forselective isolation of Gram-positive cocci). For FcMBL-based ELISAmethods, the rat blood was diluted and subjected to FcMBL-basedcolorimetric ELISA described in Example 10, where the FcMBL magneticbeads captured both live and dead pathogens directly from the dilutedrat blood, and the captured matter was then incubated with HRP-FcMBLdetection reagent followed by a colorimetric readout of OD450 with TMBsubstrate. The FcMBL-based colorimetric ELISA was performed in less than1 hour.

FIG. 34A shows results of anaerobe cultures at Day 4 of the bloodcollected from the five rats developed with intra-abdominal abscesses.While Rat #1 appeared to be sicker than Rat #2 and needed to besacrified the first, the blood culture indicated that there were morebacteria present in the blood of Rat #2 than in Rat #1. Further, theblood culture method was not sensitive enough to detect bacteria presentin Rat #3, even though Rat #3 appeared to be sick 48 hours after theimplantation.

In contrast, as shown in FIG. 34B, the FcMBL-based ELISA assay provideda better correlation of the pathogen load (including live and deadpathogens/microbial matter) with morbidity ranking than what wasindicated by blood cultures. FIG. 34C shows a substantially linearcorrelation of pathogen load determined by the ELISA using FcMBLmagnetic microbeads with morbidity ranking. Further, the FcMBL-basedELISA assay was more sensitive than the blood culture method, asevidenced by detectable levels of pathogen loads using FcMBL-based ELISAassay, as compared to undetectable levels in blood cultures, even after4 days of culturing (FIG. 34B).

A similar rat animal study was performed separately, as described above.Rats were scarified at different time points according to theirmorbidity after the implantation and their morbidity ranking is shown inTable 2.

TABLE 2 Morbidity ranking of rats after implantation of cecal contentspelvic region of rats. Morbidity ranking Sacrifice time point (scale1-5) (time after implantation) Rat #21 4-5 (with 5 the least sickest) 5days Rat #22 4-5 5 days Rat #23 1 (the most sickest) 11 hours Rat #244-5 5 days Rat #25 2 11 hours

Similar to the previous experiment, as shown in FIG. 34D, rats withpositive blood culture died of sepsis and they also had high levels ofmicrobes (live and dead) and microbial matter (e.g., endotoxin andmicrobial debris) detected by FcMBL-based ELISA. Based on FIGS. 34B and34D, the surviving rats had about 2 logs less microbes (live and dead)and microbial matter (e.g., endotoxin and microbial debris) in the bloodthan the rats which died from sepsis. The FcMBL-based ELISA wassensitive enough to detect such low levels of microbes and microbialmatter in surviving rat blood, which was usually not detectable by bloodcultures.

Accordingly, this Example shows that FcMBL microbeads can bind cecalmicrobes used in the intraabdominal sepsis model. Further, an ELISAusing FcMBL reagents can be used to rapidly detect live microbes and/ornon-viable microbial matter (including dead microbes and endotoxins) ina blood sample (e.g., 1-hour ELISA assay vs. 4-day blood culture).Further, the ELISA using FcMBL reagents is demonstrated to be moresensitive than blood cultures.

Example 18 Microbe Depletion Using FcMBL-Coated Magnetic Microbeads ofDifferent Sizes

To assess the performance of FcMBL-coated magnetic microbeads ofdifferent sizes to capture a microbe in a test sample, FcMBL-coatedmagnetic microbeads were produced by conjugating a saturating amount ofbiotinylated FcMBL molecules to magnetic microbeads of different sizes:(1) 1 μm MYONE™ T1 Streptavidin microbeads; (2) 128 nm Ademtechmicrobeads coated with streptavidin; and (3) 50 nm Miltenyi microbeadscoated with anti-biotin IgG. Appropriate volumes (e.g., ˜20 μL) ofdifferent sized FcMBL-coated magnetic microbeads were then added toaliquots of a sample (e.g., ˜1 mL) containing E. coli or S. aureuscells. The mixture was then mixed for about 10 mins (e.g., using aHULAMIXER™ sample mixer), followed by magnetic separation of themicrobeads. The supernatant after removal of the microbeads was thenplated on LB agar, which was then incubated overnight at −37° C. Anymicrobes that were not captured by the FcMBL-coated magnetic microbeadswill grow on LB agar overnight. FIG. 35 indicates successful depletionof microbes (e.g., E. coli or S. aureus) present in a test sample usingFcMBL-coated magnetic microbeads of different sizes.

All patents and other publications identified in the specification andexamples are expressly incorporated herein by reference for allpurposes. These publications are provided solely for their disclosureprior to the filing date of the present application. Nothing in thisregard should be construed as an admission that the inventors are notentitled to antedate such disclosure by virtue of prior invention or forany other reason. All statements as to the date or representation as tothe contents of these documents is based on the information available tothe applicants and does not constitute any admission as to thecorrectness of the dates or contents of these documents.

1. A microbe-binding molecule comprising: a. at least one microbesurface-binding domain; b. a substrate-binding domain adapted fororienting the microbe surface-binding domain away from a solid substratesurface; c. at least one linker between the microbe surface-bindingdomain and the substrate-binding domain; and d. a detectable label.2-226. (canceled)
 227. The microbe-binding molecule of claim 1, whereinthe microbe surface-binding domain comprises a carbohydrate recognitiondomain derived from at least one carbohydrate-binding protein selectedfrom the group consisting of lectin, collectin, ficolin, mannose-bindinglectin (MBL), maltose-binding protein, arabinose-binding protein,glucose-binding protein, Galanthus nivalis agglutinin, peanut lectin,lentil lectin, DC-SIGN, C-reactive protein, and any combinationsthereof.
 228. The microbe-binding molecule of claim 227, wherein themicrobe surface-binding domain comprises a carbohydrate recognitiondomain derived from mannose-binding lectin (MBL), wherein thecarbohydrate recognition comprises an amino acid sequence selected fromSEQ ID NOs: 1-4.
 229. The microbe-binding molecule of claim 1, whereinthe detectable label is selected from the group consisting of biotin, afluorescent dye or particle, a luminescent or bioluminescent marker, aradiolabel, an enzyme, a microbial enzyme substrate, a quantum dot, animaging agent, and any combinations thereof.
 230. The microbe-bindingmolecule of claim 229, wherein the enzyme causes a color change in thepresence of an enzyme substrate.
 231. The microbe-binding molecule ofclaim 230, wherein the enzyme is a horseradish peroxidase or alkalinephosphatase.
 232. The microbe-binding molecule of claim 1, wherein thelinker comprises a portion of a Fc region of an immunoglobulin.
 233. Themicrobe-binding molecule of claim 232, wherein the portion of the Fcregion comprises a hinge region, a CH2 region, a CH3 region, or anycombinations thereof.
 234. The microbe-binding molecule of claim 1,wherein the substrate-binding domain comprises at least one oligopeptidecomprising an amino acid sequence of Alanine-Lysine-Threonine (AKT).235. An assay comprising: a. contacting a test sample withmicrobe-binding molecules, wherein the microbe-binding molecules areconjugated to a surface of a solid substrate, and wherein the engineeredmicrobe-binding molecules each comprises: i. at least one microbesurface-binding domain; ii. a substrate-binding domain adapted fororienting the microbe surface-binding domain away from the solidsubstrate surface; and iii. at least one linker between the microbesurface-binding domain and the substrate-binding domain; and b.detecting binding of a microbe or microbial matter to at least a portionof the microbe-binding molecules.
 236. The assay of claim 235, whereinthe detecting is performed by spectroscopy, electrochemical detection,polynucleotide detection, fluorescence anisotropy, fluorescenceresonance energy transfer, electron transfer, enzyme assay, magnetism,electrical conductivity, isoelectric focusing, chromatography,immunoprecipitation, immunoseparation, aptamer binding, filtration,electrophoresis, use of a CCD camera, immunoassay, ELISA, Gram staining,immunostaining, microscopy, immunofluorescence, western blot, polymerasechain reaction (PCR), RT-PCR, fluorescence in situ hybridization,sequencing, mass spectroscopy, or any combination thereof.
 237. Theassay of claim 235, further comprising contacting the bound microbe ormicrobial matter with a labeling molecule prior to the detecting step.238. The assay of claim 235, wherein the detecting further comprisesidentifying the microbe or microbial matter bound to the microbe-bindingmolecules based on a microbe signature determined by mass spectrometricanalysis.
 239. The assay of claim 238, further comprising, prior to themass spectrometric analysis, detaching the bound microbe or microbialmatter from at least a portion of the microbe-binding molecules. 240.The assay of claim 239, wherein the detaching comprises incubating themicrobe-binding molecules comprising the bound microbe or microbialmatter in a buffer (i) having an acidic pH or (ii) comprising an ionwhich forms a salt with Ca²⁺ ion, and wherein the salt is insoluble inthe buffer.
 241. The assay of claim 235, wherein the surface of thesolid substrate further comprises at least one reference area forcomparison with a readout signal determined upon contact of the testsample with the microbe-binding molecules.
 242. The assay of claim 235,further comprising subjecting the bound microbe to at least oneantibiotic.
 243. The assay of claim 242, further comprising evaluating aresponse of the bound microbe to the at least one antibiotic.
 244. Theassay of claim 243, further identifying a treatment of an infectioncaused by the microbe.
 245. A kit comprising: a. one or more containerscontaining a population of microbe-binding microparticles; wherein theengineered microbe-binding microparticles each comprises on its surfaceat least one microbe-binding molecule, wherein the at least onemicrobe-binding molecule comprises i. at least one microbesurface-binding domain; ii. a substrate-binding domain adapted fororienting the microbe surface-binding domain away from the solidsubstrate surface; and iii. at least one linker between the microbesurface-binding domain and the substrate-binding domain; and b. one ormore containers containing a population of labeling molecules, whereinthe labeling molecules are conjugated to an enzyme; and c. one or morecontainers containing an enzyme substrate that produces a color changein the presence of the enzyme.